3-Halo cephalosporins

ABSTRACT

7-Acylamido- and 7-amino-3-halo-3-cephem-4-carboxylic acids, esters and pharmaceutically-acceptable salts and esters thereof are new cephalosporin compounds provided by the reaction of a 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester or a 7-amino-3-hydroxy-3-cephem-4-carboxylic acid ester with an iodinating, fluorinating, chlorinating, or brominating reagent. For example, 7-amino-3-chloro-3-cephem-4-carboxylic acid and 7-phenoxyacetamido-3-chloro-3-cephem-4-carboxylic acid are provided. The 7-acylamido-3-halo cephalosporin acids and pharmaceutically acceptable salts and esters provided are valuable antibiotic compounds having desirable therapeutic properties.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division, of application Ser. No. 457,150, filedApr. 1, 1974 now U.S. Pat. No. 3,962,227 which was acontinuation-in-part application of co-pending application Ser. No.335,414 filed Feb. 23, 1973, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a novel class of cephalosporin antibiotics. Inparticular it relates to 3-halo cephalosporins represented by thefollowing generalized formula ##STR1## wherein R is hydrogen or an acylgroup derived from a carboxylic acid, R₁ is hydrogen, a carboxylic acidprotecting ester group or a pharmaceutically acceptable ester or salt,and X represents fluoro, chloro, bromo, or iodo.

The compounds of the invention possess the unique structural charactericof a halogen atom directly bonded to the carbon atom in the 3-positionof the dihydrothiazine ring. According to the cepham nomenclature systemthe above depicted compounds are named as 7-amino- or7-acylamido-3-halo-3-cephem-4-carboxylic acids, salts and esters.

Prior to this invention 3-bromomethyl-3-cephem-4-carboxylic acid esters,U.S. Pat. Nos. 3,647,788 and 3,688,203, and3-bromomethyl-2-cephem-4-carboxylic acid esters, U.S. Pat. No. 3,637,678were described. These known 3-bromomethyl compounds are described asuseful intermediates for preparing cephalosporin antibiotics. Incontrast with these intermediates, the 3-halo-3-cephem-4-carboxylicacids described herein are especially valuable antibiotics.

SUMMARY OF THE INVENTION

U.S. Pat. No. 3,275,626 discloses certain7-acylamido-3-exomethylenecepham-4-carboxylic acids and esters. Inapplication Ser. No. 278,668 an electrolytic reduction process for thepreparation of 3-exomethylene cepham compounds is provided. In myco-pending application Ser. no. 118,941, filed Feb. 25, 1971, U.S. Pat.No. 3,932,393 issued Jan. 13, 1976 another process for preparing3-exomethylenecepham-4-carboxylic acids and esters is described.According to the described process a3-thiosubstituted-methyl-3-cephem-4-carboxylic acid or ester is reducedto effect the reductive displacement of the 3-thiosubstituent and form a3-exomethylenecepham-4-carboxylic acid or ester. The 7-amino- and7-acylamido-3-exomethylenecepham-4-carboxylic acid esters are convertedto the corresponding 3-hydroxy-3-cephem-4-carboxylic acid esters asdescribed in my copending application Ser. No. 310,191, filed Nov. 28,1972, U.S. Pat. No. 3,917,587 issued Nov. 4, 1975.

According to the present invention, the 7-amino-, and7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid esters are halogenatedunder moderate conditions to provide a 7-amino-, or7-acylamido-3-halo-3-cephem-4-carboxylic acid ester. By employingreadily removable ester groups well known in the cephalosporin art, the3-halo esters provided are then converted to the free acids by knownprocedures for removing such ester groups. The7-amino-3-halo-3-cephem-4-carboxylic acids provided herein can beacylated to provide the 7-acylamido-3-halo-3-cephem-4-carboxylic acidantibiotics. The 7-acylamido-3-halo-3-cephem-4-carboxylic acids providedeither by the direct halogenation of a7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester followed by esterremoval or by the acylation of a 7-amino-3-halo-3-cephem-4-carboxylicacid or ester are valuable antibiotic compounds which can be used toinhibit the growth of microorganisms pathogenic to animal and plantlife.

It is an object of this invention to provide a novel class ofcephalosporin antibiotics. In particular it is an object of thisinvention to provide 7-acylamido-3-halo-3-cephem-4-carboxylic acidantibiotic compounds. It is a further object of this invention toprovide the 3-halo substituted cephalosporin nucleus, the7-amino-3-halo-3-cephem-4-carboxylic acids as well as the esters andsalts thereof. A still further object of the present invention is toprovide a process for the preparation of the 7-amino- and7-acylamido-3-halo-3-cephem-4-carboxylic acids described herein.

DETAILED DESCRIPTION

The 3-halo cephalosporin compounds provided by this invention arerepresented by the following general formula: ##STR2## wherein R ishydrogen or an acyl group derived from a carboxylic acid and representedby the formula ##STR3## wherein R' is C₁ -C₆ alkyl, C₁ -C₃ haloalkyl, C₁-C₃ cyanoalkyl, phenyl, methylphenyl, hydroxyphenyl, halophenyl,nitrophenyl, aminophenyl, methoxyphenyl, 5-amino-5-carboxybutyl, or a5-substituted-amino-5-carboxybutyl ester group of the formula ##STR4##wherein A is diphenylmethyl, p-nitrobenzyl, benzyl,2,2,2-trichloroethyl, t-butyl, or p-methoxybenzyl and A' is C₂ -C₄alkanoyl, C₂ -C₄ haloalkanoyl, benzoyl, halobenzoyl, 2,4-dinitrophenyl,or phthaloyl;

or R' is a group of the formula ##STR5## wherein a an a' independentlyare hydrogen, C₁ -C₄ lower alkyl, C₁ -C₄ lower alkoxy, halogen, hydroxy,nitro, amino, or carboxy;

Z is O or S; and

m is 0 or 1;

or R' is a group of the formula ##STR6## wherein P is 2-thienyl,3-thienyl, phenyl or a substituted phenyl group of the formula ##STR7##wherein a and a' are as defined above, Q is hydroxyl, formyloxy,acetoxy, carboxy, or sulfo;

or R' is a group of the formula ##STR8## wherein P has the same meaningsas defined above and Y is hydrogen, methyl or acetyl;

or R' is a group of the formula

    R"--CH.sub.2 --

wherein R" is 2-thienyl, 3-thienyl, 2-furyl, 2-oxazyl, 2-thiazyl, or1-tetrazyl,

or R' is a group of the formula ##STR9## wherein a, a', Z and m have thesame meanings as defined above; R₁ is hydrogen, benzyl, 4-methoxybenzyl,4-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, t-butyl or apharmaceutically acceptable ester of the formula ##STR10## lower alkyl;and X is fluoro, chloro, bromo, or iodo, and when R₁ is hydrogen thepharmaceutically acceptable salts thereof.

In the foregoing definition of the compounds provided by this inventionthe term "C₁ -C₆ alkyl" refers to the straight and branched chain alkylhydrocarbon groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, n-amyl, isoamyl, n-hexyl, and the like; "C₁ -C₃ cyanoalkyl"refers to such groups as cyanomethyl, 2-cyanoethyl, 3-cyanopropyl, and2-cyanopropyl; "C₂ -C₄ alkanoyl" refers to acetyl, propionyl, butyryl,and the like; "C₂ -C₄ haloalkanoyl" refers to chloroacetyl, bromoacetyl,2-chloropropionyl, 3-bromobutyryl, and the like; "C₁ -C₄ lower alkyl"refers to the straight and branched chain lower alkyl hydrocarbon groupssuch as methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, and thelike; "C₁ -C₄ lower alkoxy" refers to methoxy, ethoxy, isopropoxy,n-butoxy, and the like. As used herein the term "halogen" refers tofluoro, chloro, bromo, and iodo. The term "halobenzoyl" refers to thechloro and bromo substituted benzoyl groups such as 4-chlorobenzoyl,4-bromobenzoyl, 2,4-dichlorobenzoyl, and the like.

Illustrative of the groups in the above definition represented by thefollowing formula where m is 0 are ##STR11## phenylacetyl,4-methylphenylacetyl, 3-ethylphenylacetyl, 4-isopropylphenylacetyl,2-methylphenylacetyl, 4-chlorophenylacetyl, 4-nitrophenylacetyl,4-bromophenylacetyl, 2,4-dichlorophenylacetyl, 3-bromphenylacetyl,4-iodophenylacetyl, 2-fluorophenylacetyl, 3,4-dihydroxyphenylacetyl,4hydroxyphenylacetyl, 3-hydroxyphenylacetyl, 2,6-dimethoxyphenylacetyl,3-carboxyphenylacetyl, 4-aminophenylacetyl, 3-ethoxyphenylacetyl,4-methoxyphenylacetyl, 3,4-dimethoxyphenylacetyl,4-t-butoxyphenylacetyl, 2-carboxyphenylacetyl,3-chloro-4-methylphenylacetyl, 3-nitrophenylacetyl, and the like. Whenin the above formula m = 1 and Z represents --O--, illustrative groupsare the following. Phenoxyacetyl, 4-hydroxyphenoxyacetyl,3-hydroxyphenoxyacetyl, 4chlorophenoxyacetyl, 3-bromophenoxyacetyl,3-ethylphenoxyacetyl, 4-methylphenoxyacetyl,3-hydroxy-3-methylphenoxyacetyl, 4-aminophenoxyacetyl,3-nitrophenoxyacetyl, 2-carboxyphenoxyacetyl, 2-chlorophenoxyacetyl,4-t-butylphenoxyacetyl, 4-methoxyphenoxyacetyl,3,4-dimethoxyphenoxyacetyl, 2-aminophenoxyacetyl,4-isopropoxyphenyloxyacetyl, 4-nitrophenoxyacetyl, and like acyl groups.When in the foregoing formula m = 1 and Z represents --S--, illustrativegroups are the following. Phenylmercaptoacetyl,4-chlorophenylmercaptoacetyl, 3-hydroxyphenylmercaptoacetyl,3,4-dimethylphenylmercaptoacetyl, 4-aminophenylmercaptoacetyl,3,4-dichlorophenylmercaptoacetyl, 3-bromophenylmercaptoacetyl,4-fluorophenylmercaptoacetyl, 2,6-difluorophenylmercaptoacetyl,4-nitrophenylmercaptoacetyl, 3-fluorophenylmercaptoacetyl, and likegroups.

When in the formula I R' is a 5-substituted-amino-5-carboxybutyl group,##STR12## is representative of esterified amino-protected adipoyl groupswherein the ester group is diphenylmethyl, p-nitrobenzyl, benzyl,p-methoxybenzyl, 2,2,2-trichloroethyl, or t-butyl, and the substitutedamino groups can be acetamido, propionamido, chloroacetamido, benzamido,2,4-dichlorobenzamido, 4-bromobenzamido, phthalimido, 2,4-dinitroanilinoand the like.

When in formula I R' represents a group of the formula ##STR13##illustrative acyl groups, ##STR14## are the mandeloyl group of theformula ##STR15## the O-formyl derivative thereof represented by thefollowing formula ##STR16## the α-carboxyphenylacetyl group representedby the following formula ##STR17## the α-sulfophenylacetyl grouprepresented by the formula ##STR18## as well as those 2-thienyl and3-thienyl acyl groups wherein the above formula the phenyl group isreplaced with a 2-thienyl or 3-thienyl ring.

When R' is a group of the formula in the syn or anti form ##STR19##illustrative acyl groups include ##STR20##

Illustrative of the foregoing acyl groups are 4-methylmandeloyl,4-hydroxymandeloyl, 3-hydroxymandeloyl, 4-aminomandeloyl,3-bromomandeloyl, 4-chloromandeloyl, 3-methyl-4-fluoromandeloyl,2-fluoromandeloyl, 4-fluoromandeloyl, 4-methoxymandeloyl,3,4-dimethyl-O-formylmandeloyl, 4-chloro-O-formylmandeloyl,3-amino-O-formylmandeloyl, 3-bromo-O-formylmandeloyl,3,4-dimethoxy-O-formylmandeloyl, O-acetyl mandeloyl, O-acetyl4-hydroxymandeloyl, α-carboxy-4-methylphenylacetyl,α-carboxy-3,4-dichlorophenylacetyl, α-carboxy-4-hydroxyphenylacetyl,α-carboxy-2-methoxyphenylacetyl, α-carboxy-4-isopropoxyphenylacetyl,α-carboxy-3-hydroxyphenylacetyl, α-carboxy-4-aminophenylacetyl,α-sulfo-4-methylphenylacetyl, α-sulfo-3,4-dichlorophenylacetyl,α-sulfo-4-chlorophenylacetyl, α-sulfo-4-hydroxyphenylacetyl,α-sulfo-3-methoxyphenylacetyl, α-oximino-4-hydroxyphenylacetyl,α-oximino-3-chlorophenylacetyl, α-oximino-4-carboxyphenylacetyl,α-methoximino-4-methylphenylacetyl,α-methoximino-3,5-dichlorophenylacetyl,α-methoximino-4-hydroxyphenylacetyl, α-methoximino-2-aminophenylacetyl,α-acetyloximinophenylacetyl, α-acetyloximino-2-thienylacetyl,α-acetyloximino-4-hyroxyphenylacetyl, α-oximino-2-thienylacetyl,α-carboxy-2-thienylacetyl, α-carboxy-3-thienylacetyl,α-methoximino-2-thienylacetyl, α-hydroxy-2-thienylacetyl,α-hydroxy-3-thienylacetyl, α-sulfo-2-thienylacetyl,α-formyloxy-2-thienylacetyl, α-acetoxy-2-thienylacetyl andα-methoximino-2-thienylacetyl.

When in the foregoing formula R' represents a group of the formulaR"--CH₂ --, illustrative of the acyl groups of the formula I are thefollowing: 2-thienylacetyl, 3-thienylacetyl, 2-furylacetyl,oxazyl-2-acetyl, thiazyl-2-acetyl, and the tetrazyl-1-acetyl grouprepresented by the following formula ##STR21##

When in Formula I, R' represents the group ##STR22## illustrative of theacyl groups, ##STR23## represented thereby are N-(phenylacetimidoyl)aminoacetyl, N-(phenoxyacetimidoyl)aminoacetyl,N-(phenylmercaptoacetimidoyl)aminoacetyl,N-(4-chlorophenylmercaptoacetimidoyl)aminoacetyl,N-(4-methoxyphenylacetimidoyl)aminoacetyl,N-(2,6-dimethoxyphenylacetimidoyl)aminoacetyl,N-(4-hydroxyphenoxyacetimidoyl)aminoacetyl,N-(4-chlorophenoxyacetimidoyl)aminoacetyl,N-(4-nitrophenylacetimidoyl)aminoacetyl,N-(3,4-dimethylphenylacetimidoyl)aminoacetyl,N-(4-fluorophenoxyacetimidoyl)aminoacetyl, and like mono anddisubstituted groups.

A preferred group of 3-halo cephalosporin antibiotics of this inventionare represented by the following formula II, ##STR24## wherein a, a', Zand m have the same meanings as defined above and X is chloro.Illustrative of these preferred compounds are the following.

7-phenylacetamido-3-chloro-3-cephem-4-carboxylic acid,

7-phenoxyacetamido-3-chloro-3-cephem-4-carboxylic acid,

7-(4-hydroxyphenylacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(4-chlorophenoxyacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(4-methoxyphenoxyacetamido)-3-chloro-3-cephem-4-carboxylic acid, andthe pharmaceutically acceptable esters and salts thereof.

Another preferred group of compounds represented by the formula I arethose represented by the following formula III. ##STR25## wherein R"represents 2-thienyl, 3-thienyl, 2-furyl, and 1-tetrazyl and Xrepresents chloro.

Illustrative of the foregoing preferred compounds represented by theformula III are the following.

7-(2-thienylacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(2-furylacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(3-thienylacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(1-tetrazylacetamido)-3-chloro-3-cephem-4-carboxylic acid, and thepharmaceutically acceptable esters and salts thereof.

A further preferred group of compounds represented by the formula I arethose represented by the following formula IV. ##STR26## wherein Prepresents phenyl or a substituted phenyl group as defined in formula Iand Q is hydroxy or carboxy. Illustrative of the preferred compoundsrepresented by formula IV are the following.

7-D-mandelamido-3-chloro-3-cephem-4-carboxylic acid,

7-D-(4-chloromandelamido)-3-chloro-3-cephem-4-carboxylic acid,

7-D-(4-hydroxymandelamido)-3-chloro-3-cephem-4-carboxylic acid,

7-D-(4-methoxymandelamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(α-carboxyphenylacetamido)-3-chloro-3-chloro-3-cephem-4-carboxylicacid, and the pharmaceutically acceptable esters and salts thereof.

The 3-halo cephalosporins described herein and represented by theformula I are prepared either by the direct halogenation of a7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester or by theacylation of a 7-amino-3-halo-3-cephem-4-carboxylic acid or esterthereof (formula I, R=H). The 7-amino-3-halo-3-cephem-4-carboxylic acidor ester is prepared either by the direct halogenation of thecorresponding 7-amino-3-hydroxy ester or by the cleavage of the7-acylamido side chain of a 7-acylamido-3-halo cephem ester.

The compounds represented by the formula I wherein X is chloro or bromoare prepared by reacting a 7-acylamido 3-hydroxy-3-cephem ester or a3-hydroxy-3-cephem nucleus ester in dimethylformamide (DMF) with areactive chloro or bromo compound which forms with DMF the chloro orbromo dimethyliminium chloride or bromide as represented by the formula##STR27## wherein X and X⁻ represent chloro or bromo and chloride orbromide respectively. The reactive halo iminium halide of the aboveformula is formed in situ and is a highly reactive chlorinating orbrominating intermediate. Chloro and bromo compounds which form theabove iminium halide include the commonly used chlorinating agents suchas phosgene (carbonyl chloride), oxalyl chloride, thionyl chloride, andthe phosphorus chlorides, for example, phosphorus trichloride andphosphorus oxychloride (phosphoryl chloride). Brominating reagents whichcan be employed in the present invention include carbonyl dibromde,oxalyl bromide, thionyl bromide (sulfurous oxybromide), and thephosphorus bromides, phosphorus oxybromide, and phosphorus tribromide.Phosphorus pentachloride can be employed in the preparation of the3-chloro-3-cephem compounds of the invention, however this reagentconcurrently reacts with the 7-acylamido side chain of the startingmaterial to form the imino chloride, the reactive intermediate in thewell known cephalosporin side chain cleavage reaction. Accordingly, itis preferable to use one of the other named chlorinating agents.

The chlorination and bromination of a 3-hydroxy cephem ester isconveniently carried out by employing dry DMF as the solvent. The DMF ispreferably dried over a molecular sieve before use. A co-solvent can beemployed along with excess DMF although such is not required. Forexample, co-solvent such as tetrahydrofuran dioxane, methylene chloride,dimethylacetamide or dimethyl sulfoxide can be used along with DMF. Thebrominating or chlorinating agent such as one of those enumerated aboveis desirably used in an amount corresponding to two equivalents of theamount of 3-hydroxy cephem ester starting material used. The reaction iscarried out by adding the halogenating reagent to a solution of the3-hydroxycephem ester in dry DMF maintained at a temperature of about 5°to 15° C. and allowing the reaction mixture to stand at room temperaturefor between 4 and 8 hours or longer. The reaction is initiallyexothermic and accordingly the reaction vessel is maintained in anice-water bath to maintain the temperature below about 25° C. during theinitial phase of the reaction. Thereafter the reaction mixture isallowed to stand at or about room temperature for the duration of thereaction. The extent to which the reaction has proceeded can bedetermined by thin layer chromatography.

Alternatively, the chlorination and bromination can be carried out byfirst preparing a mixture of the halogenating reagent in DMF to preformthe haloiminium halide, and then adding the mixture to a solution of the3-hydroxy-3-cephem ester in DMF, a mixture of DMF and a co-solvent or ina solvent such as dimethylacetamide or tetrahydrofuran.

The 3-chloro- or 3-bromo-3-cephem esters are recovered from the reactionproduct mixture by pouring the mixture into a water-ethyl acetatemixture and separating the organic phase containing the product. Theorganic phase is washed, dried and is evaporated to afford the3-halo-3-cephem ester as an amorphous residue. The product is obtainedcrystalline in many instances by trituration of the residue with etheror with n-hexane.

The preferred chlorinating and brominating reagents are phosphorustrichloride and phosphorus tribromide.

The 7-amino-3-halo-3-cephem-4-carboxylic acids are preferably obtainedby the cleavage of the 7-acyl group of a7-acylamido-3-halo-3-cephem-4-carboxylic acid ester followed by removalof the carboxylic acid protecting ester group.

The compounds represented by the formula I wherein X is fluoro areprepared by reacting a 7-acylamido-3-hydroxy cephem ester in an inertsolvent with the fluorinated tertiary amine,N-(2-chloro-1,1,2-trifluoroethyl)diethylamine. The fluorinated tertiaryamine is prepared by reacting diethylamine with chlorotrifluoroethyleneas described in J. Org. Chem. 29, 2187 (1964); Tetrahedron Lett. 23,1065 (1962) and Tetrahedron Lett. 26, 1249 (1962). The fluorination iscarried out by reacting the 3-hydroxy cephem ester with an equivalentamount of the fluorinated tertiary amine in an inert solvent, forexample, methylene chloride, chloroform, tetrahydrofuran, or anyunreactive compound in which the starting material and fluorinatingreagent are substantially soluble. The reaction mixture containing thestarting 3-hydroxy cephem ester and the fluorinating reagent is heatedat a temperature between about 30° and 60° C. for up to about 1 hour.

The 3-fluoro-3-cephem compounds can also be prepared as described inco-pending application Ser. No. 439,206 filed Feb. 6, 1974 now U.S. Pat.No. 3,926,978 issued Dec. 16, 1975 by reacting a sulfonate ester of a7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester with an inorganicfluoride such as potassium fluoride in the presence of a crown ether.The starting materials employed in this method, the sulfonate esters ofthe 3-hydroxy-3-cephem compounds, are prepared as described inco-pending application Ser. No. 439,207 filed Feb. 6, 1974, now U.S.Pat. No. 3,985,737 issued Oct. 12, 1976. In this method, a7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester is reacted in aninert solvent with a lower alkylsulfonyl halide or a phenyl orsubstituted phenylsulfonyl halide in the presence of a hydrogen halideacceptor to form the corresponding 3-lower alkylsulfonyloxy,3-phenylsulfonyloxy, or substituted 3-phenylsulfonyloxy derivative ofthe 3-hydroxy substitutent.

The preparation of the alkylsulfonate and arylsulfonate esters of the3-hydroxy-3-cephem esters is illustrated in the following generalizedreaction scheme. ##STR28##

In the above formulae, R represents an acyl group derived from acarboxylic acid, and R₁ has the same meanings as previously defined andW is C₁ -C₆ lower alkyl, such as methyl, ethyl, propyl, n-butyl,n-hexyl, and the like, or W is a phenyl group or a substituted phenylgroup such as methylphenyl, nitrophenyl, or halophenyl. Representativeof the sulfonyl halides which can be employed in the preparation ofthese sulfonate esters are methanesulfonyl chloride, toluenesulfonylchloride, p-fluorobenzene sulfonyl chloride, ethanesulfonyl chloride,butanesulfonyl chloride, and the like.

The reaction is carried out in an inert solvent at a temperature betweenabout -5° and 35° C., and preferably between about 15° and 25° C. Inertsolvents which can be employed in the sulfonation reaction are solventswhich are unreactive with the sulfonyl halide generally aproticsolvents. Solvents which can be employed include amides such asN,N-dimethylformamide and N,N-dimethylacetamide, and ethers such astetrahydrofuran and dioxane. A preferred solvent for the sulfonationreaction is N,N-dimethylacetamide. The reaction is carried out in thepresence of a hydrogen halide acceptor, for example, any of the commonlyemployed tertiary amines, such as pyridine and triethylamine; alkyleneoxides also can be employed, with the most preferred hydrogen halideacceptor being propylene oxide. The tertiary amine hydrogen halideacceptors are less desirable than the alkylene oxides since the doublebond in the Δ³ position of the cephem ring is susceptible toisomerization to the Δ² position in the presence of these amines.However, should isomerization to the Δ² isomer occur during sulfonateester formation, the double bond can be isomerized back to the Δ³position by oxidizing the Δ² isomer of the sulfonate ester to thesulfoxide with a peracid, such as m-chloroperbenzoic acid or peraceticacid. During sulfoxide formation the double bond shifts from the Δ² tothe Δ³ position. The sulfoxide then can be reduced in accordance withwell known methods, for example, with phosphorus trichloride, to providethe 3-cephem sulfonate ester.

A preferred sulfonate ester for the conversion of the3-hydroxy-3-cephem-4-carboxylic acid esters to the corresponding3-fluoro-3-cephem compounds is the methylsulfonate ester (mesylate).Another preferred ester is that formed with p-toluenesulfonyl chloride(tosylate ester). A preferred C₄ carboxylic acid protecting ester groupin the above described process is the p-nitrobenzyl ester.

The alkyl sulfonate or arylsulfonate esters of a 3-hydroxy-3-cephemester can be converted to the corresponding 3-fluoro-3-cephem ester bythe following general reaction scheme. ##STR29##

As indicated hereinabove, the sulfonate ester starting materials aredefined as having in the 3-position the substituent ##STR30## Thesubstituent W, as used herein, defines C₁ -C₆ lower alkyl, phenyl,tolyl, halophenyl, or nitrophenyl. Preferably, W is methyl, phenyl, orp-tolyl. Typical of the sulfonate ester groups thereby defined aremethylsulfonyloxy, ethylsulfonyloxy, n-propylsulfonyloxy,isopropylsulfonyloxy, n-butylsulfonyloxy, isobutylsulfonyloxy,t-butylsulfonyloxy, n-amylsulfonyloxy, isoamylsulfonyloxy,t-amylsulfonyloxy, n-hexylsulfonyloxy, isohexylsulfonyloxy,2,2-dimethylbutylsulfonyloxy, 3,3-dimethylbutylsulfonyloxy,2,3-dimethylbutylsulfonyloxy, 3-methylpentylsulfonyloxy,phenylsulfonyloxy, p-tolylsulfonyloxy, m-tolylsulfonyloxy,o-tolylsulfonyloxy, o-nitrophenylsulfonyloxy, m-nitrophenylsulfonyloxy,p-nitrophenylsulfonyloxy, p-chlorophenylsulfonyloxy,m-bromophenylsulfonyloxy, p-fluorophenylsulfonyloxy,o-chlorophenylsulfonyloxy, p-bromophenylsulfonyloxy, and the like.

Crown ethers are recognized in the literature, see for example, R. N.Greene, Tetrahedron Letters, No. 18 (1972) pp. 1793-1796. Crown ethersare cyclic structures comprised of a chain of alternating ethylenegroups and oxygen atoms. In the process of this invention, an 18-crown-6ether is employed. The basic unsubstituted 18-crown-6 ether structure is##STR31## Alternatively, it is named1,4,7,10,13,16-hexaoxacyclooctadecane. The common designation"18-crown-6" defines the total number of atoms in the ring (18) and thetotal number of oxygens (6) in the ring. Other 18-crown-6 ethers can beemployed in the process of this invention. These include, for example,dibenzo-18-crown-6 having the formula and dicyclohexyl-18-crown-6 havingthe formula ##STR32##

The source of fluorine for use in displacement of the sulfonate esterfunction is an inorganic fluoride of the formula M⁺ F⁻ in which M⁺refers to a sodium, potassium or silver ion. Preferably, the fluoridesalt which is employed is potassium fluoride.

The conversion of the 3-sulfonyloxy-3-cephem to the 3-fluoro-3-cephem iscarried out in an inert solvent, specifically a nitrile or anitroalkane, preferably acetonitrile, propionnitrile, nitromethane, ornitroethane, and, most preferably, acetonitrile, under substantiallyanhydrous conditions at a temperature of from about -20° C. to about+25° C., and, preferably, from about +15° C., to about +25° C. The3-sulfonate ester cephalosporin present in the selected solvent is mixedwith preferably, a maximum of one equivalent of the inorganic fluoridesalt based on the sulfonate ester. Preferably, an equivalent amount ofthe sulfonate ester and the fluoride salt are employed. The crown etherpreferably should be present in the reaction mixture in an amountequivalent on a molar basis to the amount of fluoride salt which ispresent. An excess of the crown ether can be employed; however, theexcess will serve no useful purpose and will constitute simply a wasteof valuable reagent. The reaction mixture is maintained at reactiontemperature for a period generally of from about 30 minutes to about 3hours, total time generally being dependent upon the temperature ofreaction.

The product which is obtained from the reaction will correspondstructurally in every respect to the sulfonate ester starting materialwith the exception that the group -O-SO₂ -W will have been displaced bya fluorine atom.

As mentioned hereinbefore, the initial 3-sulfonate ester cephalosporinreactant can be either a Δ³ -cephalosporin, a Δ² -cephalosporin, or amixture of the two. The active reactant is the Δ³ -cephalosporin.However, under the conditions of reaction, any Δ² -cephalosporin whichmay be present is isomerized to the corresponding Δ³ -cephalosporin, andthus the active reactant is formed in situ from any Δ² -cephalosporinwhich may be present initially in the reaction mixture.

The 3-iodo-3-cephem-4-carboxylic acids and esters of the Formula I areprepared by reacting a 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acidester in DMF with a diiodo triaryl phosphite for exampleiodotriphenoxyphosphonium iodide or a triaryl phosphate methiodide forexample triphenyl phosphate methiodide respectively represented by theformulae ##STR33## The iodination is carried out in dry DMF at about20°-35° C. by employing a molar excess of the iodinating reagent. The3-hydroxy-3-cephem ester and the iodinating agent are allowed to reactfor about 12 to 15 hours and thereafter the reaction mixture is pouredinto a mixture of ethyl acetate-water. The organic layer is separatedand is washed with a 5% solution of sodium thiosulfate to remove anyfree iodine present. Following a water wash the organic phase is driedand evaporated. The residue is triturated with ether or n-hexane toobtain the 3-iodo-3-cephem ester.

The iodinating reagents described above are known reagents which havepreviously been employed for converting hydroxylic compounds to iodocompounds, for example, as described in J. Am. Chem. Soc. 86, 2093(1964), J. Chem. Soc. (1953) 2224 and J. Chem. Soc. (1954) 2281.

The 3-iodo-7-amino-3-cephem-4-carboxylic acid, the 3-iodo nucleus(formula I, R=H, X=I), is prepared by the cleavage of the 7-acyl groupof a 7-acylamido-3-iodo-3-cephem-4-carboxylic acid ester followed byremoval of the ester group. The 7-acyl removal is effected by the knownphosphorus pentachloride in pyridine method.

As previously mentioned the 3-chloro or 3-bromo cephalosporin compoundsof the invention can be prepared either by halogenating a7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester or by acylating a3-halo-7-amino-3-cephem-4-carboxylic acid or ester. When the formermethod for preparing the compounds of the invention is chosen, the7-acylamido group of the starting material is desirably one which doesnot react with the halogenating agent under the conditions describedabove. For example, when the acyl moiety in the 7-position of thestarting material contains reactive functional groups such as thecarboxyl group, the amino group, the sulfonic acid (sulfo) group and thelike, such groups are blocked by the formation of an unreactivederivative thereof prior to the halogenation reaction described above.Illustrative of the 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acidesters which can be halogenated without concurrent halogenation in theside chain are the compounds represented by the formula I wherein R isC₂ -C₇ alkanoyl, C₂ -C₃ haloalkanoyl, C₂ -C₃ cyanoalkanoyl,phenoxyacetyl, 2-thienylacetyl, 3-thienylacetyl, and 2-furylacetyl.

The 7-amino-3-halo-3-cephem compounds of the Formula I, R=H, are bestprepared by the cleavage of the 7-acylamido side chain from a7-acylamido-3-halo-3-cephem-4 carboxylic acid ester by the well knownphosphorus pentachloride cleavage reaction. For example,7-[2-(2-thienyl)-actamido]-3-chloro-3-cephem-4-carboxylic acidp-nitrobenzyl ester is reacted with phosphorus pentachloride inmethylene chloride in the presence of pyridine to provide the iminochloride derivative thereof. The imino chloride intermediate is reactedwith an alcohol, for example methanol or isobutanol, to provide thecorresponding imino ether derivative. Hydrolysis of the imino etherprovides the p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylatehydrochloride.

The starting materials employed in the preparation of the compounds ofthe present invention are prepared as described in my co-pendingapplication Ser. No. 310,191 filed Nov. 28, 1972, U.S. Pat. No.3,917,587 issued Nov. 4, 1975. As described therein a7-acylamido-3-exomethylenecepham-4-carboxylic acid ester or a7-amino-3-exomethylenecepham-4-carboxylic acid ester is reacted withozone in an inert solvent at a temperature between -80 and 0° C. to formthe ozonide derivative of the 3-exomethylene double bond. The ozonideintermediate, which is not isolated, is decomposed by reacting theozonide in situ with a mild reducing agent such as sodium bisulfite, orpreferably sulfur dioxide, to provide the corresponding3-hydroxy-3-cephem-4-carboxylic acid ester.

The ozonolysis of a 7-amino-3-exomethylenecepham-4-carboxylic acid esteror a 7-acylamido-3-exomethylenecepham-4-carboxylic acid ester of thefollowing Formula V is carried out by passing ozone through a solutionof the 3-exomethylenecepham ester in an inert solvent at a temperaturebetween about -80° and 0° C. The exomethylene double bond reacts withozone to form in situ an intermediate ozonide which is decomposed, ashereinafter described, to form the 3-hydroxy-3-cephem ester of theFormula VI. ##STR34## In the above formulae, R is hydrogen or an acylgroup derived from a carboxylic acid and which acyl group isnon-oxidizable under the described ozonolysis conditions. R₁ is an esterforming group and preferably one which is easily removed underhydrogenolysis, or acid or base hydrolysis conditions.

Although the 3-exomethylene cephalosporins can also undergo oxidationwith ozone to form the sulfoxide, under the described ozonizationconditions the exo double bond reacts preferentially with ozone to formthe ozonide. The formation of the sulfoxide occurs as a result of overoxidation. Whereas the exo double bond reacts rapidly with ozone, thereaction at the sulfur atom of the dihydrothiazine ring to form thesulfoxide occurs at a much slower rate. However, the following overoxidation products can be formed in the ozonolysis reaction. ##STR35##

Ozone gas is prepared by means of an ozone generator of the typecommonly used in synthetic and analytical chemical work to produce ozoneby the action of an electric discharge on oxygen. One such ozonegenerator is that manufactured by the Welsback Corporation. The ozone isgenerated in a stream of oxygen which is then passed directly into thereaction vessel. The percentage of ozone contained in the oxygen streamcan be varied as desired, for example, by varying the rate of flow ofoxygen through the ozonizer as well as by varying the intensity of theelectric discharge. The percentage of ozone in the oxygen stream can bedetermined iodometrically by titrating with sodium thiosulfate theamount of iodine liberated from a standard solution of potassium iodideby ozone from the generator. The percentage of ozone in the oxygenstream is not critical, however for convenience in carrying out theozonolysis method of this invention an estimate of the amount of ozoneflowing into the reaction mixture enables one to determine the time atwhich the desired reaction should be complete and thus minimizes theformation of over oxidation products.

Alternatively, the ozonolysis reaction can be followedchromatographically. For instance, a small aliquot of the reactionmixture is withdrawn, the ozonide decomposed, and the amount ofunreacted starting material and 3-hydroxy-3-cephem product present inthe sample is assessed by a comparison of the thin layer chromatogramwith that of a known amount of starting material and 3-hydroxy-3-cephemcompound.

Inert solvents which can be used in the ozonolysis are those solvents inwhich the 3-exomethylene cepham esters are at least partially solubleand which are unreactive with ozone under the described conditions.Commonly used organic solvents such as methanol, ethanol, ethyl acetate,methyl acetate, and methylene chloride are satisfactory.

The concentration of the starting material in the inert solvent is notcritical and it is preferred to use a solvent volume sufficient to forma complete solution.

The preferred temperature in the ozonolysis reaction is between about-80° and -50° C.

When ozonide formation is complete as determined by either methoddescribed above, any excess ozone present in the reaction mixture ispurged from the mixture by bubbling nitrogen or oxygen through themixture.

Following the removal of any excess ozone, the ozonide is decomposed byadding to the reaction mixture a mild reducing reagent selected from thegroup consisting of sodium bisulfite, sulfur dioxide, and trimethylphosphite to provide the 3-hydroxy-3-cephem-4-carboxylic acid ester. Thedecomposition is carried out by adding an excess of the reducing reagentand then stirring the reaction mixture at a temperature of about -80° to0° C. until the reaction mixture is negative in the potassiumiodide-starch test.

A preferred reagent for decomposing the intermediate ozonide is gaseoussulfur dioxide. This reagent is preferred since it is completelyvolatilized from the reaction mixture during the subsequent work-up andthus does not complicate the recovery of the reaction product.

The 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid esters arerecovered from the reaction mixture by first evaporating the mixture todryness and thereafter extracting the product from the residue.Alternatively, N-acylated 3-hydroxy-3-cephem esters can be recoveredfrom the organic liquid phase of the decomposition mixture by separatingthe liquid phase from insolubles, and after washing and drying, theorganic layer is evaporated to yield the 3-hydroxy ester.

The 3-hydroxy nucleus ester, a 7-amino-3-hydroxy-3-cephem-4-carboxylicacid ester, is best isolated in the form of a salt as for example, thehydrochloride or hydrobromide salt.

When an ester of 7-amino-3-exomethylenecepham-4-carboxylic acid (FormulaV, R=H) is ozonized it is preferable to use a salt of this nucleus, forexample, the hydrochloride or p-toluenesulfonate salt.

In a specific example of the preparation of a 3-hydroxy-3-cephem ester,p-methoxybenzyl 7-phenoxyacetamido-3-exomethylenecepham-4-carboxylate isdissolved in ethyl acetate and is reacted with ozone at a temperature ofabout -78° C. The excess ozone is expelled by bubbling oxygen throughthe cold solution. The ozonide is decomposed by adding excess sodiumbisulfite to the reaction mixture at 0° C. with stirring. The organiclayer is decanted from the insolubles and is washed, dried andevaporated to yield p-methoxybenzyl7-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate.

In a further example, p-nitrobenzyl7-amino-3-methylenecepham-4-carboxylate hydrochloride is dissolved inmethanol and ozone is bubbled through the solution at a temperature ofabout -78° C. Excess ozone is purged from the mixture with nitrogen andthe ozonide is decomposed by bubbling sulfur dioxide through themixture. The reaction mixture is evaporated to dryness and the residue,p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylate is obtained asthe hydrochloride salt.

The starting material for the preparation of the 3-exomethylenecephamesters are prepared as described in my co-pending application Ser. No.118,941, filed Feb. 25, 1971, U.S. Pat. No. 3,932,313 issued Jan. 13,1976. As described therein, a 7-acylamido cephalosporanic acid isreacted with a sulfur containing nucleophile according to knownprocedures to effect the nucleophilic displacement of the acetoxy groupof the cephalosporanic acid and provide a7-acylamido-3-thiosubstituted-methyl-3-cephem-4-carboxylic acid. The3-thiosubstituted cephem product is then reduced with hydrogen in thepresence of Raney nickel or with zinc/formic acid in the presence ofdimethylformamide to produce the 3-exomethylenecepham acid. For example,7-phenylacetamidocephalosporanic acid is reacted with potassium ethylxanthate to yield7-phenylacetanido-3-ethoxythionocarbonylthiomethyl-3-cephem-4-carboxylicacid which on reduction with zinc/formic acid in the presence of DMFyields 7-phenylacetamido-3-exomethylenecepham-4-carboxylic acid of theformula ##STR36## Likewise, there is described the 3-exomethylenecephamnucleus of the formula which can be prepared by reacting a7-acylamido-3-exomethylenecepham- 4-carboxylic acid ester withphosphorus pentachloride (PCl₅) in methylene chloride in the presence ofpyridine to obtain the intermediate imino chloride. The imino chlorideis reacted with methanol in the cold to afford the imino ether. Theimino ether readily undergoes hydrolysis to provide the7-amino-3-exomethylenecepham-4-carboxylic acid ester hydrochloride. Theester group is then removed to yield the 3-exomethylenecepham nucleus.

As described above the compounds of the invention as represented by theformula I can be prepared by the acylation of a7-amino-3-halo-3-cephem-4-carboxylic acid or an ester thereof.

The acylation of these nuclei can be carried out by the known methodsused for the acylation of 7-aminocephalosporanic acid or7-aminodeacetoxycephalosporanic acid. The cephalosporin 3-halo nucleusacids or esters (Formula I, R=H) can be acylated under anhydrousacylation methods as well as in the presence of water. Accordingly, thecephalosporin-3-halo nucleus free acid or an ester thereof can beacylated with a carboxylic acid halide in an aqueous solvent system, forexample aqueous acetone, in the presence of a hydrogen halide acceptorsuch as propylene oxide, pyridine or sodium bicarbonate. The acylationcan also be effected by reacting an ester of the halo nucleus with acarboxylic acid in the presence of a condensing agent such asN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) ordicyclohexylcarbodiimide. Also the 3-halo nucleus ester can be acylatedin a mixed anhydride reaction. By yet another known acylation method,the halo nucleus can be acylated with an active ester of a carboxylicacid, for example the pentachlorophenyl ester of a carboxylic acid.

For example, p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate isreacted with phenylacetyl chloride in cold aqueous acetone containingsodium bicarbonate to provide p-nitrobenzyl7-phenylacetamido-3-chloro-3-cephem-4-carboxylate.

Diphenylmethyl 7-amino-3-bromo-3-cephem-4-carboxylate is reacted withphenoxacetyl chloride in the presence of pyridine to providediphenylmethyl 7-phenoxyacetamido-3-bromo-3-cephem-4-carboxylate.

p-Methoxybenzyl 7-amino-3-chloro-3-cephem-4-carboxylate is reacted withmandelic O-carboxy-anhydride in ethyl acetate to provide p-methoxybenzyl7-(D-α-mandelamido)-3-chloro-3-cephem-4-carboxylate.

Illustrative of these derivatives of the acyl groups R'-C=O which can beemployed in the acylation of the halo nucleus esters of free acidsprovided by this invention or the following: thiophene-2-acetylchloride, phenoxyacetyl chloride, phenylacetyl chloride,oxazole-2-acetyl bromide, thiazole-2-acetyl chloride, tetrazole-1-aceticacid, mandelic acid O-carboxy anhydride, 4-hydroxymandelic acidO-carboxy anhydride, 4-chlorophenoxyacetyl bromide, benzoyl chloride,2,6-dimethoxybenzoyl chloride, thiophene-3-acetyl chloride,furyl-2-acetyl chloride, the pentachlorophenyl ester of phenylmercaptoacetic acid, 4-chlorophenylmercaptoacetyl chloride, and like acylatingreagents.

Illustrative of the antibiotic cephalosporins of the Formula I whereinR' is represented by R"CH₂ -- are the following:

7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid,

7-[2-(2-thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylic acid,

7-[2-(3-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid,

7-[2-(2-furyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid,7-[2-(2-furyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid,

7-[2-(2-oxazyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid,

7-[2-(2-thiazyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid,

7-[2-(2-thiazyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid,

7-[2-(1-tetrazyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid,

7-[2-(1-tetrazyl)acetamido]-3-fluoro-3-cephem-4-carboxylic acid, and thebenzyl, diphenylmethyl, m-methoxybenzyl, p-nitrobenzyl,2,2,2-trichloroethyl and tert-butyl esters and the pharmaceuticallyacceptable esters and non-toxic base addition salts thereof.

The compounds of the Formula I wherein Q is a carboxylic acid group canbe prepared for example, by the acylation of a 3-halo-3-cephem nucleuswith tert-butyl phenylmalonic acid chloride, or with tert-butyl2-thienylmalonic acid chloride in the presence of a hydrogen halideacceptor such as sodium bicarbonate. For example, tert-butyl7-amino-3-chloro-3-cephem-4-carboxylate is reacted with 2 equivalents oftert-butyl phenylmalonyl chloride in acetone at a temperature of about5° C. and in the presence of excess sodium bicarbonate to provide,tert-butyl7-(α-tert-butyloxycarboxylphenylacetamido)-3-chloro-3-cephem-4-carboxylate.Removal of both tert-butyl ester groups with 90% formic acid providesthe diacid, 7-(α-carboxyphenylacetamido)-3-chloro-3-cephem-4-carboxylicacid.

Illustrative compounds represented by the Formula I wherein Q is acarboxy group are7-(α-carboxyphenylacetamido)-3-chloro-3-cephem-4-carboxylic acid,7-[2-carboxy-2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylicacid,7-(α-carboxy-4-hydroxyphenylacetamido)-3-chloro-3-cephem-4-carboxylicacid, 7-(α-carboxyphenylacetamido)-3-fluoro-3-cephem-4-carboxylic acid,7-[2-carboxy-2-(2-thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylicacid, 7-(α-carboxyphenylacetamido)-3-bromo-3-cephem-4-carboxylic acid,7-(α-carboxy-4-chlorophenylacetamido)-3-chloro-3-cephem-4-carboxylicacid,7-(α-carboxy-3,4-dimethoxyphenylacetamido)-3-chloro-3-cephem-4-carboxylicacid,7-[2-carboxy-2-(3-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylicacid,7-(α-carboxy-3-hydroxyphenylacetamido)-3-chloro-3-cephem-4-carboxylicacid and the benzyl, diphenylmethyl, p-methoxybenzyl, p-nitrobenzyl,2,2,2-trichloroethy and tert-butyl esters and the pharmaceuticallyacceptable non-toxic base addition salts thereof.

The α-sulfo-acylamido-3-halo-cephem compounds of the Formula I wherein Qis a sulfo group (--SO₃ H) are prepared by following the acylationprocedures employed for the preparation of α-sulfobenzylpenicillinsdescribed by U.S. Pat. No. 3,660,379 and in the J. Med. Chem., 15 (11),11-5 (1972); ibid, p. 1108. For example,7-amino-3-chloro-3-cephem-4-carboxylic acid is reacted withα-sulfophenylacetyl chloride in a mixture of acetone and watercontaining an excess of sodium bicarbonate to yield7-(α-sulfophenylacetamido)-3-chloro-3-cephem-4-carboxylic acid. Examplesof α-sulfoacylamido-3-halo-cephems of the present invention are,

7-(α-sulfo-3-chlorophenylacetamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(α-sulfo-4-hydroxyphenylacetamido)-3-chloro-3-cephem-4-carboxylicacid,

7-(α-sulfo-4-methoxyphenylacetamido)-3-bromo-3-cephem-4-carboxylic acid,

7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid,

7-[2-sulfo-2-(3-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid,

7-[2-sulfo-2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid,

7-[2-sulfo-2-(2-thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylic acid,

7-(α-sulfo-3,4-dimethylphenylacetamido)-3-chloro-3-cephem-4-carboxylicacid and the benzyl, diphenylmethyl, p-methoxybenzyl, p-nitrobenzyl,2,2,2-trichloroethyl and tert-butyl esters and the pharmaceuticallyacceptable non-toxic base addition salts thereof.

The compounds of the Formula I wherein R is an acyl group of the formula##STR37## are prepared by the acylation of a 3-halo-3-cephem-nucleusester with an α-methoximino or α-acetyloximino glyoxamoyl chloridewherein Y is methyl or acetyl. The α-oximino compounds, Y=H, areprepared by the base hydrolysis of the α-acetyloximino compounds. Forexample, p-nitrobenzyl7-(α-methoximinophenylglyoxamido)-3-chloro-3-cephem-4-carboxylate isprepared by the acylation of p-nitrobenzyl7-amino-3-chloro-3-cephem-4-carboxylate withα-methoximinophenylglyoxamoyl chloride in an acetone-water mixturecontaining an excess of sodium bicarbonate.

p-Nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate is acylated withα-acetytoximinophenylglyoxamoyl chloride in acetone in the presence ofpyridine to yield p-nitrobenzyl7-(α-acetyloximinophenylglyoxamido)-3-chloro-3-cephem-4-carboxylate.

The α-oximino compounds, Y=H, are obtained by the mild base hydrolysisof the α-acetyloximino compounds. For example, p-nitrobenzyl7-(α-acetyloximinophenylglyoxamido)-3-chloro-3-cephem-4-carboxylate isreacted in aqueous acetone for 12 hours at room temperature with onemole of sodium hydroxide to yield p-nitrobenzyl7-(α-oximinophenylglyoxamido)-3-chloro-3-cephem-4-carboxylate.

The foregoing is illustrated by the following reaction scheme whichdepicts arbitrarily the syn form. ##STR38## wherein P, R₁ and X have thesame meanings as previously defined.

Illustrative of the 7-(α-oximino, α-acetyloximino andα-methoximinoarylglyoxamido-3-halo cephalosporins provided by thisinvention are

7-(α-oximinophenylglyoxamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(α-methoxyimino-4-chlorophenylglyoxamido)-3-chloro-3-cephem-4-carboxylicacid,

7(α-oximino-4-hydroxyphenylglyoxamido)-3-chloro-3-cephem-4-carboxylicacid,

7-[α-oximino-2-(2-thienyl)glyoxamido]-3-chloro-3-cephem-4-carboxylicacid,

7-(α-oximinophenylglyoxamido)-3-fluoro-3-cephem-4-carboxylic acid,

7-(α-methoximinophenylglyoxamido)-3-bromo-3-cephem-4-carboxylic acid,

7-[α-oximino-2-(3-thienyl)glyoxamido]-3-chloro-3-cephem-4-carboxylicacid,

7-[α-oximino-2-(2-thienyl)glyoxamido]-3-fluoro-3-cephem-4-carboxylicacid

7-(α-acetyloximinophenylglyoxamido)-3-chloro-3-cephem-4-carboxylic acid,

7-(α-acetyloximinophenylglyoxamido)-3-fluoro-3-cephem-4-carboxylic acid,

7-[α-acetyloximino-2-(2-thienyl)glyoxamido]-3-chloro-3-cephem-4-carboxylicacid, and the benzyl, diphenylmethyl, p-methoxybenzyl, p-nitrobenzyl,2,2,2-trichloroethyl and tertbutyl esters and the pharmaceuticallyacceptable esters and non-toxic base addition salts thereof.

The antibiotic compounds of the Formula I, wherein R' is a substitutedimidoyl aminoacetamido group of the formula ##STR39## are prepared witha 7-(3-benzyl, 3-phenoxymethyl, or3-phenylmercaptomethyl-1,2,4-oxadiazole-5-one-4-acetamido)-3-halo-3-cephem-4-carboxylicacid by hydrogenation in the presence of Raney nickel catalyst atneutral pH by following the method described in U.S. Pat. No. 3,669,958.The following reaction scheme is illustrative ##STR40## wherein a, a',Z, m and X are as previously defined.

The substituted oxadiazole 3-halocephalosporin is prepared by acylationof a 3-halo nucleus acid or ester, for example7-amino-3-chloro-3-cephem-4-carboxylic acid, with a 3-substituted1,2,4-oxadiazole-5-one-4-acetyl chloride under conventional acylationconditions such as those described in U.S. Pat. No. 3,669,958.

Alternatively these intermediates can be prepared by reacting a7-haloacetamido-3-halo-3-cephem-4-carboxylic acid or ester (Formula I,R=chloro or bromoacetyl) with the 3-substituted 1,2,4-oxadiazole-5-onein the presence of a hydrogen halide acceptor such as pyridine.

Illustrative of the 7-substituted-imidoyl aminoacetamido-3-halocephalosporin antibiotics represented by the Formula are,

7-[N-(phenoxyacetimidoyl)aminoacetamido]-3-chloro-3-cephem-4-carboxylicacid,

7-[N-(phenylacetimidoyl)aminoacetamido]-3-chloro-3-cephem-4-carboxylicacid,

7-[N-(phenylmercaptoacetimidoyl)aminoacetamido]-3-chloro-3-cephem-4-carboxylicacid,

7-[N-(4-chlorophenylmercaptoacetimidoyl)aminoacetamido]-3-chloro-3-cephem-4-carboxylicacid,

7-[N-(phenylacetimidoyl)aminoacetamido]-3-fluoro-3-cephem-4-carboxylicacid,

7-[N-(phenoxyacetimidoyl)aminoacetamido]-3-bromo-3-cephem-4-carboxylicacid,

7-[N-(4-hydroxyphenoxyacetimidoyl)aminoacetamido]-3-chloro-3-cephem-4-carboxylicacid,

7-[N-(4-chlorophenoxyacetimidoyl)aminoacetamido]-3-chloro-3-cephem-4-carboxylicacid,

7-[N-(4-nitrophenylacetimidoyl)aminoacetamido]-3-chloro-3-cephem-4-carboxylicacid, and the benzyl, diphenylmethyl, p-methoxybenzyl, p-nitrobenzyl,2,2,2-trichloroethyl and tertbutyl esters and the pharmaceuticallyacceptable esters and non-toxic base addition salts thereof.

Especially useful compounds of this invention are the 3-halo nucleusesters and acids of the Formula I wherein R is hydrogen. The 3-halonucleus esters and acids are represented by the following formula##STR41## wherein X and R₁ are as previously defined. When R₁ ishydrogen, the zwitterionic form of the compound can exist as illustratedbelow. ##STR42##

Another especially useful group of compounds provided herein arerepresented by the Formula I wherein R is 5-amino-5-carboxyvaleryl or anesterified amino-protected 5-amino-carboxyvaleryl group represented bythe formula ##STR43## wherein A and A' are as previously defined. Suchcompounds are represented by the following formula ##STR44## wherein Xand R₁ are as defined previously.

The above represented compounds are valuable intermediates for thepreparation of the 3-halo nucleus acids and esters in that they canundergo the well known 7-acylamido side-chain cleavage reaction withphosphorus pentachloride and pyridine by following the cleavageprocedures by which 7-aminocephalosporanic acid esters are prepared withcephalosporin C.

They are prepared with cephalosporin C in the following manner.Initially, the side-chain amino group and both carboxyl groups ofcephalosporin C are protected with such groups as A and A' and then theprotected molecule is reacted with a sulfur containing nucleophile, forexample, potassium ethyl xanthate or thiourea to form the3-thio-substituted methyl derivative by nucleophilic displacement of theacetoxy function of the 3-acetoxymethyl group. Thereafter the3-thio-substituted methyl-3-cephem ester is reduced with either Raneynickel in the presence of hydrogen or zinc/formic acid in the presenceof DMF to effect the reductive displacement of the 3-thio substituentand provide the 3-exomethylenecepham ester. The above reaction sequenceis described in my co-pending application Ser. No. 118,941, filed Feb.25, 1971, U.S. Pat. No. 3,932,393 issued Jan. 13, 1976.

The 3-exomethylenecepham ester is then reacted with ozone and theozonide decomposed to form the 3-hydroxy-3-cephem ester according to themethod described previously. The 3-hydroxy ester is then halogenatedaccording to the method of this invention to provide a compound of theabove formula VIII.

Cleavage of the protected 7-acyl side-chain with PCl₅pyridine/isobutanol or methanol affords the 3-halo nucleus ester of theformula VII. Cleavage of the 7-acyl group can also be carried out withnitrosyl chloride by the method described by U.S. Pat. No. 3,188,311,when the amino adipoyl group is unprotected.

Illustrative of the compounds represented by the above formula VIII are

diphenylmethyl7-[5-diphenylmethyloxycarbonyl-5-(2,4-dichlorobenzamido)valeramido]-3-chloro-3-cephem-4-carboxylate,

diphenylmethyl7-(5-diphenylmethyl-5-chloroacetamidovaleramido)-3-chloro-3-cephem-4-carboxylate,

p-nitrobenzyl7-(5-p-nitrobenzyloxycarbonyl-5-propionamidovaleramido)-3-chloro-3-cephem-4-carboxylate,

diphenylmethyl7-[5-diphenylmethyloxycarbonyl-5-(4-chlorobenzamido)valeramido]-3-chloro-3-cephem-4-carboxylate,

p-nitrobenzyl7-(5-p-nitrobenzyloxycarbonyl-5-acetamidovaleramido)-3-chloro-3-cephem-4-carboxylateand

diphenylmethyl7-[5-diphenylmethyloxycarbonyl-5-(2,4-dichlorobenzamido)valeramido]-3-bromo-3-cephem-4-carboxylate.

Examples of the 7-amino-3-halo-3-cephem acids and esters which areprovided are the following.

7-amino-3-chloro-3-cephem-4-carboxylic acid,

7-amino-3-bromo-3-cephem-4-carboxylic acid,

7-amino-3-fluoro-3-cephem-4-carboxylic acid,

7-amino-3-iodo-3-cephem -4-carboxylic acid,

diphenylmethyl 7-amino-3-chloro-3-cephem-4-carboxylate,

p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate,

p-nitrobenzyl 7-amino-3-bromo-3-cephem-4-carboxylate,

p-methoxybenzyl 7-amino-3-fluoro-3-cephem-4-carboxylate,

2,2,2-trichloroethyl 7-amino-3-chloro-3-cephem-4-carboxylate,

benzyl 7-amino-3-bromo-3-cephem-4-carboxylate and the like.

The term "pharmaceutically acceptable" esters as used herein refers tothe C₁ -C₄ straight and branched chained alkanoyloxymethyl esters suchas acetoxymethyl, propionyloxymethyl and pivaloyloxymethyl esters. Theseesters of the 7-acylamido-3-halo-3-cephem-4-carboxylic acids possessantibiotic activity at levels akin to those of the free acid form of thecephalosporin compound and can be used in place of the antibiotic in thefree acid form. In contrast, the other ester groups within thedefinition of R₁, namely the benzyl, benzhydryl, p-methoxybenzyl,p-nitrobenzyl, 2,2,2-trichloroethyl and t-butyl esters, do not possessany significant antibiotic acitivity.

The 7-acylamido-3-halo-3-cephem-4-carboxylic acids (Formula I, R═R'-C═O,R₁ ═H) are useful antibiotic compounds for combating infectionsattributable to gram-positive and gram-negative organisms. The compoundscan be administered by injection (s.c. or i.m.) in the free acid form orin the form of a pharmaceutically acceptable ester or non-toxic acidaddition salt. Salts formed with the free acids and inorganic bases suchas sodium bicarbonate, potassium carbonate, sodium hydroxide and calciumhydroxide provide the sodium, potassium and calcium salts which can beformulated for administration for example, as isotonic solutions or asliquid suspensions.

The pharmaceutically acceptable esters, for example the acetoxymethylesters, are prepared by reacting a7-acylamido-3-halo-3-cephem-4-carboxylic acid alkali metal salt such asthe potassium salt with the alkanoyloxymethyl halide such asacetoxymethyl chloride in aqueous acetone.

The in vitro antimicrobial activity of the 3-halo cephalosporins isillustrated by the data in the following tables. Table I contains theantimicrobial activity for7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid obtainedin the standard Disc-plate method. The numerical values are the diameterin millimeters of the zones of inhibition observed with the namedorganisms.

                  TABLE I                                                         ______________________________________                                        ANTIBIOTIC ACTIVITY OF                                                        7-[2-(2-Thienyl)acetamido]-                                                   3-chloro-3-cephem-4-carboxylic acid                                                         Zone of Inhibition                                                            (diameter in mm)                                                              Concentration (mg/ml)                                           Test Organism   1.0      0.1       0.01                                       ______________________________________                                        Staphylococcus aureus                                                                         31       29        20                                         Bacillus subtilis                                                                             48       40        24                                         Sarcina lutea   37       28        21                                         Mycobacterium avium                                                                           14       --        --                                         Proteus vulgaris                                                                              17       12H.sup.1 --                                         Salmonella gallinarum                                                                         32       22        --                                         Escherichia coli                                                                              24       19        Tr.sup.2                                   Klebsiella pneumoniae                                                                         36       27        13                                         Pseudomonas solanacearcum                                                                     28       21        --                                         ______________________________________                                         .sup.1 H = hazy zone of inhibition                                            .sup.2 Tr = trace zone of inhibition                                     

The following Table II lists the minimum inhibitory concentration (MIC)for 7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acidagainst penicillin resistant Staphylococcus both in the presence of andabsence of serum. The MIC values were obtained by the Gradient Platetechnique carried out essentially as described by Bryson and Szybalski,Science, 116, 45 (1952).

                  TABLE II                                                        ______________________________________                                        ANTIBIOTIC ACTIVITY vs.                                                       Penicillin Resistant Staphylococcus                                                       Minimum Inhibitory Concentration                                              (mcg/ml)                                                          Clinical Isolate                                                                            No Serum     Serum                                              ______________________________________                                        V 41          5.0          7.0                                                V 32          8.4          >20                                                X400.sup.1    >20          >20                                                V 84          0.8          1.0                                                X 1.1         0.2          <0.1                                               ______________________________________                                         .sup.1 Methicillin resistant Staphylococcus                              

In Table III, below, the MIC values for the test compound of Tables Iand II against representative gramnegative organisms is presented. Thedata were obtained by the Gradient Plate technique.

                  Table III                                                       ______________________________________                                        Antibiotic Activity                                                           vs.                                                                           Gram-Negative Organisms                                                                        Minimum Inhibitory Conc.                                     Test Organism    (mcg/ml)                                                     ______________________________________                                        Shigella sp.     25                                                           Escherichia coli 18.5                                                         Klebsiella pneumoniae                                                                          0.6                                                          Aerobacter aerogenes                                                                           0.8                                                          Salmonella heidelberg                                                                          0.8                                                          Pseudomonas aeruginosa                                                                         >200                                                         Serratia marcescens                                                                            >200                                                         ______________________________________                                    

In Table IV below, the in vitro antimicrobial activity of7-[2-(2-thienyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid ispresented for several illustrative microorganisms. The data presentedwere obtained in the standard disc-plate method with the numericalvalues recording the diameter in millimeters of the zones of inhibitionobserved with the named microorganisms.

                  TABLE IV                                                        ______________________________________                                        ANTIBIOTIC ACTIVITY OF                                                        7-[2-(2-THIENYL)ACETAMIDO]-                                                   3-BROMO-3-CEPHEM-4-CARBOXYLIC ACID                                                          Zone of Inhibition.sup.1                                                      (diameter in mm)                                                              Concentration (mg./ml.)                                         Test Organism   1.0      0.1      0.01                                        ______________________________________                                        Staphylococcus aureus                                                                         27       25       22                                          Bacillus subtilis                                                                             37       38       23                                          Sarcina lutea   32       33       17                                          Mycobacterium avium                                                                           23       h 42     h 33                                        Saccharomyces pastorianus                                                                     --       --       --                                          Neurospora sp.  --       --       --                                          Candida albicans                                                                              --       --       --                                          Bacillus subtilis*                                                                            60       50       43                                          Trichophyton mentagrophytes                                                                   --       --       --                                          Proteus vulgaris                                                                              28       20       h 11                                        Salmonella gallinarum                                                                         30       15       --                                          Escherichia coli                                                                              23       14       --                                          Pseudomonas aeruginosa                                                                        --       --       --                                          Klebsiella pneumoniae                                                                         28       19       11                                          Serratia marcescens                                                                           --       --       --                                          Pseudomonas solanacearcum                                                                     31       h 20     --                                          Escherichia coli*                                                                             27       18       h 11                                        ______________________________________                                         *Synthetic medium                                                             .sup.1 An h = hazy zone                                                       A dash indicates no observed zone of inhibition                          

The following Table V lists the minimum inhibitory concentration (MIC)for the antibiotic7-[2-(2-thienyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid againstpenicillin resistant strains of Staphylococcus in the absence of serum.The inhibitory concentrations were obtained by the Gradient Platemethod.

                  TABLE V                                                         ______________________________________                                        ANTIBIOTIC ACTIVITY VS.                                                       PENICILLIN RESISTANT STAPHYLOCOCCUS                                                          Minimum Inhibitory                                             Clinical Isolate                                                                             Concentration (mcg./ml.)                                       ______________________________________                                        V 41           0.5                                                            V 32           0.6                                                            X400.sup.1     >20                                                            V 84           0.4                                                            X 1.1          0.3                                                            ______________________________________                                         .sup.1 Methicillin resistant Staphylococus                               

The minimum inhibitory concentrations for7-[2-(2thienyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid againstrepresentative gram-negative bacteria is presented in Table VI. The datawere obtained by the Gradient Plate Method.

                  TABLE VI                                                        ______________________________________                                        ANTIBIOTIC ACTIVITY OF                                                        7-[2-(2-THIENYL)ACETAMIDO]-                                                   3-BROMO-3-CEPHEM-4-CARBOXYLIC ACID                                            VS.                                                                           GRAM-NEGATIVE BACTERIA                                                                         Minimum Inhibitory                                           Test Organism    Concentration (mcg./ml.)                                     ______________________________________                                        Shigella sp.     19.8                                                         Escherichia coli 20.8                                                         Klebsiella pneumoniae                                                                          1.0                                                          Aerobacter aerogenes                                                                           1.0                                                          Salmonella heidelberg                                                                          1.0                                                          Pseudomonas aeruginosa                                                                         >200                                                         Serratia marcescens                                                                            >200                                                         ______________________________________                                    

The antibiotic activity of the O-formyl ester of7-(D-mandelamido)-3-chloro-3-cephem-4-carboxylic acid is demonstrated bythe data presented in the following tables. Tables VII and VIII list theminimum inhibitory concentrations of the test compound effective againstrepresentative penicillin resistant Staphylococcus strains, while TableVII lists the MIC values obtained against representative gram-negativemicroorganisms. The data were obtained by the Gradient Plate method.

                  TABLE VII                                                       ______________________________________                                        ANTIBIOTIC ACTIVITY VS.                                                       PENICILLIN RESISTANT STAPHYLOCOCCUS                                                      Minimum Inhibitory                                                            Concentration (mcg./ml.)                                           Clincal Isolate                                                                            No Serum      Serum                                              ______________________________________                                        V 41         5.8           >20                                                V 32         8.0           >20                                                 X400.sup.1  >20           >20                                                V 84         0.4           1.0                                                 X 1.1       <0.1          <0.1                                               ______________________________________                                         .sup.1 Methicillin resistant Staphylococcus                              

                  TABLE VIII                                                      ______________________________________                                        ANTIBIOTIC ACTIVITY VS.                                                       GRAM-NEGATIVE ORGANISMS                                                                        Minimum Inhibitory                                           Test Organism    Concentration (mcg./ml.)                                     ______________________________________                                        Shigella sp.     4.0                                                          Escherichia coli 6.3                                                          Klebsiella pneumoniae                                                                          1.0                                                          Aerobacter aerogenes                                                                           0.7                                                          Salmonella heidelberg                                                                          0.6                                                          Pseudomonas aeruginosa                                                                         >200                                                         Serratia marcescens                                                                            160                                                          ______________________________________                                    

In Table IX which follows, the MIC values against a spectrum ofmicroorganisms obtained with the test compound are presented. The testmethod was the standard agar dilution method.

                  TABLE IX                                                        ______________________________________                                        ANTIBIOTIC SPECTRUM OF                                                        7-[D-(O-FORMYL)MANDELAMIDO]-3-                                                CHLORO-3-CEPHEM-4-CARBOXYLIC ACID                                                                    Minimum Inhibitory                                     Test                   Concentration                                          Microorganism          (mcg./ml.)                                             ______________________________________                                        Staphylococcus aureus 3055                                                                           >0.25                                                  Staphylococcus aureus 3074                                                                           1                                                      Streptococcus faecalis X66                                                                           64                                                     Proteus morganii PR15  4                                                      Salmonella typhosa SA12                                                                              0.5                                                    Klebsiella pneumoniae KL14                                                                           4                                                      Enterobacter aerogenes EB17                                                                          128                                                    Serratia marcescens SE3                                                                              >128                                                   Escherichia coli EC14  8                                                      Citrobacter freundii   >128                                                   Pseudomonas aeruginosa X239                                                                          128                                                    Bordetella bronchiseptica 16                                                                         64                                                     Salmonella typhimurium 1                                                      Pseudomonas solanacearcum X185                                                                       128                                                    Erwinia amylovora      >128                                                   Candida tropicalis A17 >128                                                   Trichophyton mentagrophytes 27                                                                       >128                                                   Aspergillus flavus E   >128                                                   Ceratocystis ulmi      >128                                                   ______________________________________                                    

The 7-acylamido-3-halo-3-cephem-4-carboxylic acid esters ##STR45## areuseful as intermediates in the preparation of the free acid antibioticforms of the compounds. Ester forming groups within the definition of R₁are all known groups commonly employed to protect the C₄ carboxylic acidgroup of the cephalosporin molecule while reactions involving othergroups in the molecule are performed. These ester forming groups arereadily removed to provide the free acid by known reduction orhydrolysis procedures. For example, the p-nitrobenzyl ester group isremoved via catalytic hydrogenolysis over palladium on carbon (U.S. Pat.No. 3,632,850); the diphenylmethyl group (benzhydryl) is removed withtrifluoroacetic acid in anisole at about 10° C.; the p-methoxybenzylgroup is removed with trifluoroacetic acid at about 10° C. [J. Org.Chem. 36, 1259 (1971)]; the 2,2,2-trichloroethyl group is removed withzinc and acid [J. Am. Chem. Soc. 88, 852 (1966)]; the benzyl ester groupis removed via catalytic hydrogenolysis over palladium catalyst [U.S.Pat. No. 3,197,466, J. Org. Chem. 27, 1381 (1962)]; and the tert-butylgroup is removed as described in J. Org. Chem., 31, 444 (1966).

The 7-amino-3-halo-3-cephem-4-carboxylic acids and esters (Formula I, R═ H) are valuable intermediates useful in preparing the 3-haloantibiotics. As previously described these 3halo nucleus acids andesters are acylated by following conventional N-acylation procedures toprovide the 7-acylamido-3-halo-3-cephem-4-carboxylic acids or esters.

The following Examples are provided to further illustrate the compoundsof the invention and the methods and procedures employed in theirpreparation.

A. Preparation of Starting Materials

EXAMPLE 1 p-Nitrobenzyl 7-amino-3-methylenecepham-4-carboxylatehydrochloride.

To a solution of 965 mg. (2 mmole) of p-nitrobenzyl7-phenoxyacetamido-3-methylenecepham-4-carboxylate in 10 ml. ofmethylene chloride were added 175 mg of dry pyridine and 460 mg. ofphosphorus pentachloride and the mixture was stirred at room temperaturefor 6 hours. One ml. of isobutanol was added to the mixture which wasthen stored at 0° C. overnight. The reaction product, p-nitrobenzyl7-amino-3-methylenecepham-4-carboxylate hydrochloride, which formed as acrystalline precipitate was filtered to yield 430 mg. (58% yield).

Elemental Analysis for C₁₅ H₁₆ N₃ O₅ SCl Theory: C, 46.69; H, 4.18; N,10.89 Found: C, 46.40; H, 4.20; N, 10.62

I.R. (Nujol Mull) Carbonyl absorption of 5.65 (β-lactam) and 5.75(ester) microns.

N.M.R. (DMSO d₆) signals at 6.34 (2d, 2H, C₂ -H₂), 4.98 (d, 1H, C₆ -H);4.7-4.4 (m, 6H, C₄ -H, ester CH₂, C₄ -CH₂ and C₇ -H); and 2.4-1.6 (m,4H, aromatic H) tau.

EXAMPLE 2 p-Nitrobenzyl 7-amino-3-methylenecepham-4-carboxylatep-toluenesulfonate salt

To a solution of 965 mg. of p-nitrobenzyl7-phenoxyacetamido-3-methylenecepham-4-carboxylate in 10 ml. ofmethylene chloride were added 175 mg. of dry pyridine and 460 mg. ofphosphorus pentachloride and the mixture was stirred for five hours atroom temperature. Thereafter the reaction mixture was cooled to 0° C.and 50 ml. of cold methanol were added. Following a stirring period ofone-hour at room temperature the reaction mxture was evaporated in vacuoto remove the solvents and the residual reaction product mixture wasdissolved in a mixture of ethyl acetate and water. The pH was adjustedto pH 7 and the ethyl acetate layer was separated and was washed withwater and dried. One equivalent of p-toluene sulfonic acid was added tothe dried solution and on cooling 600 mg. of p-nitrobenzyl7-amino-3-methylenecepham-4-carboxylate p-toluenesulfonate formed as acrystalline precipitate. The product was purified by recrystallizationfrom a mixture of 12 ml. of methanol 24 ml. of ether and 15 ml. ofpetroleum ether.

Elemental analysis for C₂₂ H₂₃ N₃ O₈ S₂ : Theory: C, 50.66; H, 4.45; N,8.06, Found: C, 50.41; H, 4.51; N, 7.86.

I.R. (Nujol Mull) carbonyl absorption at 5.65 (β-lactam) and 5.71(ester) microns.

N.M.R. (DMSO d₆)

Signals at 7.70 (s, 3H, p-methyl); 6.39 (s, 2H, C₂ --H₂); 4.98 (d, 1H,C₆ --H); 4.7-4.3 (m, 6H C₄ --H, ester CH₂ ; C₃ --CH₂, and C₇ --H); and2.93-1.68 (m, 8H, aromatic H) tau.

U.V.(pH 6 buffer) Maxima at 219 mμ (ε=19,600) and 268 mμ (ε=9,400).

EXAMPLE 3 p-Methoxybenzyl 7-amino-3-methylenecepham-4-carboxylatehydrochloride

To a solution of 4.3 g. of p-methoxybenxyl7-phenoxyacetamido-3-methylenecepham-4-carboxylate in 50 ml. ofmethylene chloride were added 880 mg. of dry pyridine and 2.3 g. ofphosphorus pentachloride and the mixture was stirred at the refluxtemperature for 3 hours. The reaction mixture was then cooled in anice-water bath and 5 ml. of isobutanol were added. The mixture wasstirred in the cold for several hours during which time 2.2 g. of thereaction product. p-methoxybenzy 7-amino-3-methylenecepham-4-carboxylatehydrochloride, precipitated from the mixture. The product was filteredand washed with cold methylene chloride and was dried in vacuo.

Elemental analysis for C₁₆ H₁₉ N₂ O₄ SCl: Theory: C, 51.82; H, 5.16; N,7.55, Found: C, 51.65; H, 5.04; N, 7.72.

EXAMPLE 4 p-Methoxybenzyl 7-amino-3-methylenecepham-4-carboxylatep-toluenesulfonate

To a solution of 937 Mg. of p-methoxybenzyl7-phenoxyacetamido-3-methylenecepham-4-carboxylate in 10 ml. ofmethylene chloride was added 0.18 ml. of dry pyridine and 460 mg. ofphosphorus pentachloride. The mixture was stirred at room temperaturefor 2 hours and was then cooled to 5° C. To the cold mixture was added50 ml. of cold methanol and the mixture was allowed to warm to roomtemperature. The reaction mixture was evaporated in vacuo and theresidue was dissolved in a mixture of ethyl acetate and water. The pH ofthe solution was adjusted to pH 7 and the ethyl acetate layer wasseparated, washed with water and dried. To the dried ethyl acetate layerwas added one equivalent of p-toluene sulfonic acid. On cooling 600 mg.of p-methoxybenzyl 3-methylenecepham-4-carboxylate p-toluenesulfonateprecipitated as a crystalline solid.

Elemental analysis for C₂₃ H₂₆ N₂ O₆ S₂ : Theory: C, 54.53; H, 5.17; N,5.53, Found: C, 54.33; H, 5.05; N, 5.47.

I.R. (Nujol Mull): Carbonyl absorption band at 5.65 (β-lactam) and 5.78(ester) microns.

N.M.R. (DMSO d₆): Signals at 7.69 )s, 3H, para methyl) 6.41 (s, 2H, C₂--H₂) 6.23 (s, 3H, para methoxy) 5.0 (d, 1H, C₆ --H) 4.82 (s, 2H, esterCH₂) 4.7-4.55 (m, 4H, C₄ --H, C₃ --CH₂ and C₇ --H) 3.2-2.0 (m, 8H,aromatic H) tau.

EXAMPLE 5 p-Nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylatehydrochloride

A solution of 3.85 g. of p-nitrobenzyl7-amino-3-methylenecepham-4-carboxylate hydrochloride, prepared asdescribed by Example 1, in 600 ml. of methanol was cooled in anacetone-dry ice bath. Ozone was bubbled through the reaction mixture forapproximately 20 minutes at which time the reaction mixture developed afaint blue coloration. Nitrogen was then passed through the reactionmixture to expel excess ozone. Next the intermediate ozonide wasdecomposed by passing sulfur dioxide gas through the reaction mixtureuntil the mixture gave a negative potassium iodide-starch test.

The reaction mixture was evaporated in vacuo and the residue wasdissolved in 200 ml. of 0.1N hydrogen chloride in methylene chloride.The solution was evaporated to dryness and the residual reaction productwas dissolved in acetone. On cooling, 3.15 g. of p-nitrobenzyl7-amino-3-hydroxy-3-cephem-4-carboxylatehydrochloride precipitated as acrystalline solid.

I.R. (Nujol Mull): Carbonyl absorption at 5.55 (β-lactam carbonyl) and5.02 (ester carbonyl hydrogen bonded to 3 hydroxy) microns.

Electrometric titration (66% DMF) pKa 4.0 and 6.3

EXAMPLE 6 p-Nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylatehydrochloride

A solution of 4 g. of p-nitrobenzyl7-amino-3-methylenecepham-4-carboxylate hydrochloride in 620 ml. ofmethanol was cooled in a dry ice-acetone bath and ozone was bubbledthrough the cold solution for about 20 minutes. The reaction mixture waspurged of the remaining ozone by passing nitrogen through the solutionand 10 g. of sodium bisulfite were added.

The reaction mixture was stirred for one hour at ice-bath temperature atwhich time the mixture gave a negative potassium iodide starch test.

The mixture was evaporated in vacuo to yield the reaction product as anamorphous yellow residue. The residue was crystallized in acetone toyield 3.4 g. of p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylatehydrochloride as a crystalline acetone solvate.

I.R. (Nujol Mull): Carbonyl absorption bands at 5.60 (β-lactam) and 6.04(ester carbonyl hydrogen bonded to 3 hydroxy) microns.

N.M.R. (DMSO d₆): signals at 7.92 (s, 3H, 1/2 mole acetone), 6.22 (2d,2H, C₂ --H₂), 5.07 (d, 1H, C₆ H), 4.8-4.5 (m, 3H, ester CH ₂ and C₇ H),2.4-1.6 (m, 4H, aromatic H) tau.

EXAMPLE 7 p-Nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylatehydrochloride.

Following the ozonization procedure described by Examples 5 and 6, 3.85g. of p-nitrobenzyl 7-amino-3-methylenecepham-4-carboxylatehydrochloride was ozonized in methanol and the intermediate ozonide wasdecomposed at a temperature of 0° C. with 3.5 ml. of trimethylphosphite. The reaction mixture was evaporated and the residue wasdissolved in 100 ml. of 0.1N HCl in methylene chloride. The saidsolution was evaporated and the residue was crystallized from acetone toyield 2.8 g. of p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylatehydrochloride.

EXAMPLE8 p-Nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylate

Four millimole of p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylatehydrochloride, prepared as described in Example 5, was dissolved inwater and ethyl acetate was added to the solution. The pH of the slurrywas adjusted from pH 2.2 to pH 5 with 1N sodium hydroxide. The ethylacetate layer was separated and was washed with water and dried overmagnesium sulfate. The dried ethyl acetate layer was evaporated todryness to yield 1.2 g. of p-nitrobenzyl7-amino-3-hydroxy-3-cephem-4-carboxylate as a crystalline residue.

Elemental analysis for: C₁₄ H₁₃ N₃ O₆ S: Theory: C, 44.86; H, 3.73; N,11.96, Found: C, 47.87; H, 4.00; N, 12.11.

I.R. (Nujol Mull): Carbonyl absorption at 5.65 (broad, β-lactam and,ester) and 6.0 (amide) microns.

N.M.R. (DMSO d₆): signals at 6.63 (2d, 2H, C₂ H), 5.31 (d, 1H, C₆ H),4.89 (d, 1H, C₇ H), 4.62 (s, 2H, ester CH₂), 4.30 (broad s, 2H, 7 N-H),2.5-1.8 (m, 4H, aromatic H) and 1.2 (d, 1H, C₃ OH) tau.

EXAMPLE 9 Methyl 7-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate

A solution of 1.6 g. of methyl7-phenoxyacetamido-3-methylenecepham-4-carboxylate in 300 ml. ofmethylene chloride was cooled in an acetone-dry ice bath. Ozone wasbubbled through the cold solution for three minutes at which time thereaction mixture developed a slight blue coloration. Excess ozone wasexpelled with a stream of oxygen and 10 g. of sodium bisulfite wereadded. The reaction mixture was stirred and allowed to warm to 0° C. Theliquid phase was separated by decantation and was washed successivelywith a 5% solution of hydrochloric acid, water and a saturated solutionof sodium chloride. The washed mixture was dried and evaporated to yield1.5 g. of crude methyl7-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate as an amorphoussolid.

The crude product was dissolved in ethyl acetate and was extracted witha 5% solution of sodium bicarbonate. Ethyl acetate was added to theextract which was then acidified to pH 2 with 1N hydrochloric acid. Theorganic phase was separated and washed with a saturated solution ofsodium chloride and dried. The dried extract was evaporated to drynessto yield 709 mg. of the reaction product contaminated with a minoramount of the corresponding 3-hydroxy-3-cephem sulfoxide, an overoxidation product. The product was separated from the sulfoxide impurityand obtained pure by preparative thin layer chromatography on silica gelwith chloroform: methanol (9:1).

Elemental analysis for: C₁₆ H₁₆ N₂ O₆ S.H₂ O, Theory: C, 50.26; H, 4.75;N, 7.33; S, 8.38, Found: C, 51.03; H, 4.62; N, 7.06; S, 8.37.

I.R. (chloroform): absorption peaks at 2.8 (amide NH), 5.6 (β-lactamcarbonyl), 5.85 (broad, amide and, ester carbonyl and 6.6 (amide II)microns.

N.M.R. (CDCl₃): signals at 6.65 (s, 2H, C₂ -H₂), 6.13 (s, 3H, methylester), 5.40 (s, 2H, side-chain CH₂), 4.93 (d, 1H, C₆ H), 4.32 (q, 1H,C₇ H), 3.15-2.38 (m, 6H, aromatic and amide H), and 1.06 (broad s, 1H,3-OH) tau.

Electrometric titration (66% aqueous DMF): pKa5.6.

EXAMPLE 10 p-Methoxybenzyl7-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate.

A solution of 2.5 g. of p-methoxybenzyl7-phenoxyacetamido-3-methylenecepham-4-carboxylate in 350 ml. of ethylacetate was cooled in an acetone-dry ice bath. Ozone was bubbled throughthe cold solution for 8 minutes and then oxygen was passed through theozonized reaction mixture to expel excess ozone. The intermediateozonide was decomposed by adding to the reaction mixture 25 g. of sodiumbisulfite with stirring at a temperature of about 0° C. The reactionsolution was decanted and was washed successively with water, 5%hydrochloric acid and a saturated solution of sodium chloride. Thewashed mixture was dried and evaporated to yield the reaction product,p-methoxybenzyl 7-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate asan amorphous solid.

N.M.R. (CDCl₃): signals at 6.73 (s, 2H, C₂ H₂), 6.23 (s, 3H, p-methoxy),5.53 (s, 2H, side-chain CH₂), 5.03 (d, 1H, C₆ H), 4.87 (s, 2H, esterCH₂), 4.47 (g, 1H, C₇ H), 3.40-2.50 (m, 9H, aromatic H), 2.33 (d, 1H,amode NH), and 1.53 (broad s, 1H, 3 OH) tau. EXAMPLE 11

p-Nitrobenzyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate

To a solution of 1.55 g. of p-nitrobenzyl7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in 30 ml. ofacetone containing 364 mg. (0.5 ml., 3.6 mmole) of triethylamine wasadded 962 mg. of urea. With stirring at room temperature, a solution of730 mg. (4.4 mmole) of 2-thiophene acetyl chloride in 20 ml. of acetonewas added dropwise to the mixture. After 2.5 hours the reaction mixturewas filtered and evaporated. The residue was dissolved in ethyl acetateand the solution was washed successively with water, a 5% solution ofsodium bicarbonate, 5% hydrochloric acid, and a saturated solution ofsodium chloride. The washed solution was dried and then was concentratedby evaporation in vacuo to yield 1.2 g. of the reaction product as acrystalline residue. The product was recrystallized from ethyl acetateto yield pure p-nitrobenzyl 7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate having the following spectral properties.

I.R. (Nujol Mull): absorption peaks at 3.0 (amide NH), 5.68 (β-lactamcarbonyl), and 6.1 (amide, and ester hydrogen bonded to 3 OH) microns.

N.M.R. (CDCl₃ /DMSO d₆):signals at 6.54 (2d, 2H, C₂ H₂), 6.16 (s, 2H,side-chain CH₂), 4.90 (d, 1H, C₆ H) 4.60 (d, 2H, ester CH₂), 4.43 (q,1H, C₇ H), 3.1-1.6 (m, 7H, aromatic H) and 1.30 (d, 1H, amide NH) tau.

EXAMPLE 12 p-Nitrobenzyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate.

p-Nitrobenzyl 7-amino-3-methylenecepham-4-carboxylate hydrochloride,3.85 g., was reacted with ozone in methanol as described in Example 5 toprovide the ozonide. The ozonide was decomposed with sulfur dioxide toproduce the 3-hydroxy product which was isolated as crude product. Thecrude 3-hydroxy-3-cephem nucleus ester was dissolved in 175 ml. oftetrahydrofuran and 50 ml. of water. Sodium bisulfite, 2.1 g., wassuspended in the solution and a solution of 4.8 g. of 2-thiophene acetylchloride in 200 ml. of THF was added dropwise to the suspension.

The mixture was stirred for two hours at room temperature and was thenevaporated to an aqueous residue. The residue was slurried with ethylacetate, the organic layer separated and washed with 5% hydrochloricacid and with water. The washed layer was dried and evaporated todryness to yield the reaction product as a crystalline residue. Theresidue was triturated three times with diethyl ether to removecontaminating 2-thiophene acetic acid and to provide 2.9 g. of thepurified crystalline product, p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate.

Electrometric titration (66% aqueous DMF) pKa 5.9.

N.M.R. (CDCl₃ /D₂ O): signals at 6.60 (s, 2H, C₂ H₂), 6.13 (s, 2H,side-chain CH₂), 4.96 (d, 1H, C₆ H), 4.62 (d, 2H, ester CH₂), 4.46 (d,1H, C₇ H) and 3.1-1.7 (m, 7H, aromatic H) tau.

EXAMPLE 13 p-Nitrobenzyl 7-acetamido-3-hydroxy-3-cephem-4-carboxylate.

A solution of 10 mmole of p-nitrobenzyl7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in a mixture of325 ml. pf acetone and 125 ml. of water was cooled in an ice-water bath.With stirring a stream of ketone gas was bubbled through the solutionfor 30 minutes. Thereafter the reaction mixture was evaporated to removeacetone and the aqueous residue was slurried with ethyl acetate. Theethyl acetate layer was separated and was washed with 5% hydrochloricacid and a saturated solution of sodium chloride. The washed extract wasdried and evaporated in vacuo to yield the reaction product as acrystalline residue. The residue was triturated with diethyl ether andvacuum dried to yield 3.55 g. of p-nitrobenzyl7-acetamido-3-hydroxy-3-cephem-4-carboxylate melting at about 146°-152°C. with decomposition.

Elemental analysis for: C₁₆ H₁₅ N₃ O₇ S:

Theory: C, 48.85; H, 3.84; N, 10.68

Found: C, 48.97; H, 3.96; N, 10.42.

I.R. (CHCl₃): absorption bands at 2.9 and 3.0 (amide NH and OH), 5.63(β-lactam carbonyl) and 5.95 (broad, amide, and ester carbonyl hydrogenbonded to 3 OH) microns.

N.M.R. (CDCl₃): signals at 7.90 (s, 3H, 7-acetamido CH₃), 6.55 (s, 2H,C₂ H₂), 4.92 (d, 1H, C₆ H), 4.63 (m, 2H, ester CH₂), 4.30 (q, 1H, C₇ H),2.81 (d, 1H, amide NH), 2.5-1.8 (m, 4H, aromatic H), and 2.8 (s, 1H, C₃OH) tau.

Electrometric titration (66% aqueous DMF) pKa 5.9

EXAMPLE 14 p-Nitrobenzyl7-phenylacetamido-3-hydroxy-3-cephem-4-carboxylate.

Following the ozonization procedures described in Example 9, a solutionof 350 mg. of p-nitrobenzyl7-phenylacetamido-3-methylenecepham-4-carboxylate in 250 ml. ofmethylene chloride was cooled to -78° C. and was ozonized. Theintermediate ozonide was decomposed in situ with sulfur dioxide and thereaction product was recovered and obtained crystalline by extractionwith ethyl acetate.

Elemental analysis for C₂₂ H₁₉ N₃ O₇ S:

Theory: C, 56.28; H, 4.80; N, 8.95 Found: C, 56.11; H, 4.15; N, 8.74.

N.M.R. (CDCl₃): signals at 6.68 (2d, 2H, C₂ H₂), 6.37 (s, 2H, side-chainCH₂), 5.03 (d, 1H, C₆ H), 6.66 (d, 2H, ester CH₂), 4.40 (q, 1H, C₇ H),2.7 (m, 6H, amide NH and aromatic H), 2.53-1.70 (q, 4H, aromatic H) anda singlet in low field integrating for 1H of C₃ hydroxy group tau.

I.R. (Nujol Mull): absorption peaks at 3.04 (amide), 5.60 and 6.0(β-lactam, ester and amide carbonyls) microns.

EXAMPLE 15 p-Nitrobenzyl7-(D-α-phenyl-α-formyloxyacetamido)-3-hydroxy-3-cephem-4-carboxylate

To a solution of 1.54 g. of p-nitorbenzyl7-amino-3-hydroxy-3-cepehm-4-carboxylate hydrochloride in 120 ml. ofacetone and 40 ml. of water was added 936 mg. of sodium bisulfite. Withstirring a solution of 960 mg. of O-formyl-D-mandelic acid chloride in20 ml. of anhydrous acetone was added dropwise at room temperature. Thereaction mixture was stirred at room temperature for 16 hours and wasthen evaporated to remove acetone. The aqueous residue was slurried withethyl acetate and the organic layer separated. The extract was washedwith water was dried and evaporated. The crystalline residue wastriturated with diethyl ether and dried to yield 1 g. of p-nitrobenzyl7-(D-α-phenyl-α-formyloxyacetamido)-3-hydroxy-3-cephem-4-carboxylate.

Elemental analysis for C₂₃ H₁₉ N₃ O₉ S: Theory: C, 53.80; H, 3.73; N,8.18

Found: C, 53.51, H, 3,81; N, 8.46

I.R. (CHCl₃): carbonyl absorption peaks at 5.55, 5.73, 5.85 and 5.93microns.

N.M.R. (CDCl₃): signals at 6.61 (s, 2H, C₂ H₂), 4.95 (d, 1H, C₆ H), 4.61(d, 2H, ester CH₂), 4.39 (q, 1H, C₇ H), 3.70 (s, 1H, α-CH), and2.80-1.70 (m, 11H, amide NH and aromatic H) tau.

B. Preparation of 3-halo-3-cephem acids and esters.

EXAMPLE 16 Diphenylmethyl7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate.

a. To a solution of 34 g. (100 mmole) of7-[2-(2-thienyl)acetamido]-3-methylenecepham-4-carboxylic acid in 500ml. of methylene chloride was added 21.4 g. (110 mmole) of diphenyldiazomethane and the resulting mixture was stirred for 2 hours at roomtemperature. The solvent was evaporated under reduced pressure and theresidue was dissolved in ethyl acetate. The ethyl acetate solution waswashed with a 5% solution of sodium bicarbonate, then with water and wasdried over magnesium sulfate. The dried solution was concentrated to asmall volume. On standing 40 g. of diphenylmethyl7-[2-(2-thienyl)acetamido]-3-methylenecepham-4-carboxylate melting atabout 132°-133° C. precipitated as a crystalline solid.

IR (chloroform): absorption peaks at 2.9 (amide N-H), 5.65, 5.75 and5.93 (β-lactam, ester and amide carbonyls respectively) and 6.62 (amideII) microns

NMR (CDCl₃): signals at 6.72 (ABq, 2H, C₂ --H₂), 6.21 (s, 2H, α-CH₂),4.83-4.65 (m, 4H, C₄ --H, C₆ --H and C₃ --CH₂), 4.39 (q, 1H, C₇ --H),3.4-2.65 (m, 15H, C₇ --NH, ester CH and aromatic H) tau.

b. To the solution of 8.1 g. (16 mmole) of the above ester in 80 ml. ofmethylene chloride were added 1.57 g. (1.6 ml, 19.6 mmole) of drypyridine and 3.8 g. (18.1 mmole) of phosphorus pentachloride. Thereaction mixture was stirred for 2 hours at room temperature and wasthereafter cooled in an ice-water bath. The cold mixture was treatedwith 8 ml. of isobutanol with stirring. Stirring was cntinued for 2hours during which time 3 g. of diphenylmethyl7-amino-3-methylenecepham-4-carboxylate hydrochloride formed as acrystalline precipitate. The product was filtered and washed withmethylene chloride and vacuum dried.

Elemental analysis (percent) for C₂₁ H₂₁ N₂ O₃ SCl: Theory: C, 60.50; H,5.08; N, 6.72; Cl, 8.50; Found: C, 60.70; H, 5.02; N, 6.71; Cl, 8.80.

NMR (DMSO d₆): signals at 6.45 (ABq, 2H, C₂ --H₂), 5.00 (d, 1H, C₆ --H),4.68 (d, 1H, C₇ --H), 4.60 (s, 2H, 3--CH₂), 4.44 (s, 1H, C₄ --H), 3.10(s, 1H, ester CH), and 2.61 (s, 10H, aromatic H) tau.

c. the 7-amino-3-exomethylenecepham ester hydrochloride salt product,2.1 g. (5 mmole) was dissolved in 200 ml. of methanol and the solutionwas cooled in an acetone-dry ice bath. Ozone was bubbled into the coldsolution for 7 minutes to form the intermediate ozonide. The ozonide wasdecomposed by passing a stream of sulfur dioxide gas through thereaction mixture for 2 minutes. Thereafter the reaction mixture wasevaporated and the residue was triturated with diethyl ether to yield1.6 g. of diphenylmethyl 7-amino-3-hydroxy-3-cephem-4-carboxylatehydrochloride as a crystalline solid.

NMR (CDCl₃): signals at 6.4 (ABq, 2H, C₂ --H₂), 5.0-4.5 (m, 2H, C₆ --Hand C₇ --H), 3.2-2.4 (m, 11H, ester CH and aromatic H) tau.

IR (Chloroform): carbonyl absorption peaks at 5.57 and 5.70 (β-lactamand ester carbonyl respectively) microns.

UV (pH7 buffer): λ max 275 mμ, ε= 7550.

Electrometric titration (60% aq. DMF): titratable groups at 4.5 and 6.5.

d. To a solution of 840 mg. of diphenylmethyl7-amino-3-hydroxy-3-cephem-4-carboxylate in 10 ml. of water and 10 ml.of acetone was added one gram of sodium bisulfite. The mixture wasstirred and 800 mg. of thiophene-2-acetyl chloride in 10 ml. of acetonewere added dropwise. The mixture was stirred for 4.5 hours at roomtemperature and was then evaporated under reduced pressure. The residuewas dissolved in a mixture of ethyl acetate and an aqueous 5% solutionof sodium bicarbonate. The ethyl acetate layer was separated, washedwith water and dried. The dried solution was evaporated and the residuetriturated with ether to yield 500 mg. of diphenylmethyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate.

NMR (CDCl₃): signals at 6.79 (s, 2H, C₂ --H₂), 6.16 (s, 2H, α-CH₂), 5.0(d, 1H, C₆ --H), 4.32 (q, 1H, C₇ --H), 3.05-2.46 (m, 15H, C₇ --NH, esterCH and aromatic H) tau.

I.R. (chloroform): absorption peaks at 2.9 (amide NH), 5.6, 5.73 and5.95 (β-lactam, ester and amide carbonyls respectively) and 6.65 (amideII) microns.

e. To a solution of 4.2 g. of diphenylmethyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 44 ml. ofdry dimethylformamide was added 865 mg. of phosphorus trichloride. Themixture was stirred for 1.5 hours at room temperature and was pouredinto an ethyl acetate 5% aqueous hydrochloric acid mixture. The ethylacetate layer was evaporated, was washed with 5% hydrochloric acid,water and was dried. The dried solution was concentrated in vacuo andthe product crystallized. The 3-chloro ester was filtered, washed withcold ethyl acetate and dried to yield 2.2 g.

Elemental analysis (percent) for C₂₆ H₂₁ N₂ O₄ S₂ Cl: Theory: C, 59.48;H, 4.03; N, 5.34; Cl, 6.75 Found: C, 59.77, H, 4.25; N, 5.40; Cl, 6.91.

NMR (CDCl₃): signals at 6.49 (ABq, 2H, C₂ --H₂), 6.22 (s, 2H, α-CH₂),5.08 (d, 1H, C₆ --H), 4.19 (q, 1H, C₇ --H), 3.13-2.5 (m, 15H, C₇ --NH,ester CH, and aromatic H) tau.

I.R. (CHCl₃): absorption peaks at 2.9 (amide NH), 5.55, 5.72 and 5.90(β-lactam, ester and amide carbonyls) and 6.60 (amide II) microns.

U.V. (dioxane): λ max 275 mμ, ε= 8700.

EXAMPLE 17 p-Nitrobenzyl7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate (via thionylchloride)

To a solution of 1.9 g. (4 mmole) of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 10 ml. ofDMF (dried over a molecular sieve) was added 950 mg. (0.58 ml., 8 mmole)of freshly distilled thionyl chloride. The mixture was stirred at roomtemperature for 6.5 hours and was then poured into 100 ml. of ethylacetate. The mixture was extracted three times with 30 ml. portions of5% hydrochloric acid and with a saturated solution of sodium chloride.The washed ethyl acetate solution was filtered and evaporated to drynessin vacuo. The residue was triturated with ether to yield 1.2 g. ofp-nitrobenzyl 7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylateas a brown crystalline solid melting at about 164°-166° C.

Elemental analysis (percent) for C₂₀ H₁₆ N₃ O₆ S₂ Cl: Theory: C, 48.63;H, 3.27; N, 8.51; Cl, 7.18 Found: C, 48.47; H, 3.29; N, 8.78; Cl, 6.96.

IR (Chloroform) showed absorption bands at 2.9 (amide NH), 5.59(β-lactam carbonyl), 5.75 (ester carbonyl) and 5.92 microns (amidecarbonyl).

UV absorption spectrum (acetonitrile) showed maxima at λ max 235 mμ, ε =12,100 and λ max 268 mμ, ε = 15,800. The mass spectrum of the productshowed a molecular ion of 493 m/e.

NMR (CDCl₃) showed signals at 6.39 (ABq, 2H, C₂ --H₂), 6.17 (s, 2H,α-CH₂), 4.99 (d, 1H, C₆ --H), 4.64 (s, 2H, ester Ch₂), 4.19 (q, 1H, C₇--H), 3.45 (d, 1H, C₇ --NH), 3.1-1.67 (m, 7H, aromatic H) tau.

EXAMPLE 18 7-[2-(2-Thienyl)acetamido]-3-chloro-3-cephem-4-carboxylicacid

To a solution of 995 mg. (2 mmole) of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate, prepared asdescribed in Example 17, in 60 ml. of tetrahydrofuran and 100 ml. ofmethanol containing 5 drops of 1N hydrochloric acid was added one gramof 5% palladium on carbon catalyst. The catalyst was prereduced as asuspension in 40 ml. of ethanol at room temperature under 50 psihydrogen pressure prior to use.

The suspension was hydrogenated at room temperature for 2.5 hours undera hydrogen pressure of 50 psi. The catalyst was filtered and washed onthe filter with THF and with water. The combined filtrate and catalystwashes were evaporated to dryness and the reaction product residue wasdissolved in a mixture of ethyl acetate and water. The pH of thesolution was adjusted to pH 2.5 and the ethyl acetate layer wasseparated. The acid reaction product was extracted with water from theethyl acetate solution at pH 7. The aqueous phase was separated, layeredwith ethyl acetate and acidified to pH 2.5. The ethyl acetate layer wasseparated, washed with water, dried over sodium sulfate and dried invacuo. The amorphous residue was triturated with ether to yield 165 mg.of 7-[2-2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid as acrystalline solid melting at about 114°-120° C. with decomposition andsoftening at about 110° C.

The reduction product has the following physical characteristics.

I.R. (nujol mull) showed absorption bands at 3.1 (amide NH), 5.64 and5.75 (β-lactam and carboxylic acid carbonyls respectively) and 6.1(amide II) microns.

U.V. (acetonitrile): absorption maxima at λ max 235, ε = 10,700. λ max268, ε = 7200.

NMR (CDCl₃) shows signals at 6.38 (ABq, 2H, C₂ --H₂), 6.16 (s, 2H,α-CH₂), 4.98 (d, 1H, C₆ --H), 4.20 (q, 1H, C₇ --H) and 3.1-2.5 (m, 4H,aromatic H and C₇ --NH) tau.

Percent Elemental Composition for C₁₃ H₁₁ N₂ O₄ SCl: Theory: C, 43.52;H, 3.09; N, 7.81; Cl, 9.88 Found: C, 43.55; H, 3.79; N, 7.27; Cl, 9.28.

EXAMPLE 19

p-Nitrobenzyl 7-[2-(2-Thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate(via phosphorus trichloride)

To a cooled solution of 439 mg. (0.93 mmole) of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 4.4 ml.of DMF was slowly added 85 mg. (0.05 ml., 0.63 mmole) of phosphorustrichloride. The reaction mixture was allowed to stand for 4 hours atroom temperature and thereafter the reaction product mixture wasworked-up by following the recovery procedures described in Example 19to provide 374 mg. of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate. The NMRspectrum of the product was consistent with the expected product andwith that of the compound of Example 17.

EXAMPLE 20 7-Phenoxyacetamido-3-chloro-3-cephem-4-carboxylic acid

Following the chlorination procedure of Example 19 p-nitrobenzyl7-phenoxyacetamido-3-chloro-3-cephem-4-carboxylate was prepared withphosphorus trichloride. The p-nitrobenzyl ester group was removed by theacidic hydrogenolysis procedure described by Example 18 to provide the3-chlorocephalosporanic acid antibiotic compound.

EXAMPLE 21 p-Nitrobenzyl7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate (viaphosphorus oxychloride)

To a solution of 325 mg. (0.7 mmole) of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 3.3 ml.of DMF cooled in an ice-water bath, was slowly added 212 mg. (0.13 ml.,1.4 mmole). The mixture was allowed to stand for 4 hours at roomtemperature and 225 mg. of the product was recovered by following thework-up procedures described in Example 17. The nuclear magneticresonance spectrum of the product was consistent with the spectrum ofthe previously characterized compound.

EXAMPLE 22 p-Nitrobenzyl7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate (via oxalylchloride)

To a solution of 439 mg. (0.93 mmole) of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 4.4 ml .of DMF cooled in an ice bath was added dropwise 118 mg. (0.07 ml., 0.93mmole) of oxalyl chloride. The reaction mixture was allowed to stand for4 hours at room temperature and was then poured into a mixture ofaqueous 5% hydrochloric acid and ethyl acetate. The organic layer wasseparated and was washed sequentially with 5% hydrochloric acid, waterand a saturated solution of sodium chloride. The washed layer was driedand evaporated to dryness to yield the reaction product, p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate as anamorphous solid. The product was obtained crystalline by triturating theamorphous residue with ether. Yield 360 mg. The infrared spectrum andNMR spectrum of the crystalline product were consistent with the spectraof authentic material.

EXAMPLE 23 7-[2-(2-Thienyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid

To a solution of 19 g. (40 mmole) of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-2-hydroxy-3-cephem-4-carboxylate in 300 ml ofdry DMF was added 15 g. (56mmole) of phosphorus tribromide and thereaction mixture was stirred at room temperature overnight. The reactionmixture was poured into a mixture of ethyl acetate and water and theorganic phase was separated and washed repeatedly with water and driedover magnesium sulfate. The dried organic phase was evaporated in vacuoto dryness. The crude reaction product residue weighing about 9 g. waspurified by chromatography over 500 g. of silica gel using ethylacetate-hexane (55:45 v:v) as eluent. The eluate was evaporated todryness under reduced pressure and the product, p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-bromo-3-cephem-4-carboxylate was obtainedcrystalline by triturating the dry residue with diethyl ether.

U.V. (ethanol) λ max, 270 mμ (μ=13,300), and λ max. 243 mμ (μ=12,700).

Elementary analysis calculated for C₂₀ H₁₆ BrN₃ O₆ S₂ : Theory: C,44.61; H, 3.00; N, 7.81; Br, 14.84. Found: C, 44.78; H, 3.03; N, 7.65;Br, 14.91.

Nuclear magnetic resonance spectrum (DMSO d6) showed signals at at 6.21(s, 2H, α--CH₂), 5.98 (ABq, 2H, C₂ --H₂), 4.72 (d, 1H, C₆ --H), 451 (s,2H, ester--CH₂), 420 (q, 1H, C₇ --H), 3.04-1.74 (m, 7H; aromatic H) and0.66 (d, H, C₇ --CH) tau.

The above 3-bromo ester was de-esterified in the following manner. Theester, 545 mg. (1.0 mmole) was hydrogenated at room temperature inethanol in the presence of pre-reduced 5 percent palladium-on-carboncatalyst. The catalyst was filtered and the filtrate evaporated underreduced pressure to dryness. The residual product was triturated withdiethyl ether to yield 180 mg. (44 percent) of crystalline product,7-[2-(2-thienyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid.

Electrometric titration (66 percent aqueous DMF) showed a pKa of 4.4 andan apparent molecular weight of 393. The calculated molecular weight =403.

Elemental analysis calculated for C₁₃ H₁₁ BrN₂ O₄ S₂. 1/2 diethyletherate: Theory: C, 40.91; H, 3.66; N, 6.36; Br, 18.15. Found: C,41.29; H, 3.20; N, 6.29; Br, 18.50. Nuclear magnetic resonance spectrum(CDCl₃) showed signals at 8.8 (t, diethyl ether-CH₃), 6.68-5.86 (m, C₂--H₂, α-CH₂ and diethyl ether-CH₂), 4.90 (d, 1H, C₆ -H), 3.0-2.63 (m,3H, aromatic-H), and 1.9 (d, 1H, amide NH) tau.

EXAMPLE 24

Following the bromination method of Example 23, p-nitrobenzyl7-phenoxyacetamido-3-bromo-3-cephem-4-carboxylate is prepared withphosphorus tribromide.

EXAMPLE 25

Following the bromination method of Example 23, 2,2,2-trichloroethyl7-acetamido-3-bromo-3-cephem-4-carboxylate is prepared with phosphorustribromide.

EXAMPLE 26

7-[2-(2-Thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylic acid.

To a solution of 325 mg. (0.7 mmole) of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 10 ml. ofmethylene chloride was slowly added an equivalent amount ofN-(2-chloro-1,1,2-trifluoroethyl) diethylamine. The reaction mixture washeated for 30 minutes under gentle reflux and was then evaporated todryness in vacuo. The residue was dissolved in a mixture of ethylacetate-water and the organic layer was separated. The organic layer waswashed with 5% hydrochloric acid, water and brine and was then dried.The dried reaction product solution was concentrated to a small volumeand n-hexane was added to precipitate, p-Nitrobenzyl7-[2-(2-thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylate.

The ester group was removed by catalytic hydrogenolysis by the methoddescribed in Example 18 to yield the 3-fluoro cephalosporin free acidantibiotic.

EXAMPLE 27 7-[2-(2-Thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylicacid

To a solution of 4.75 g. (10 mmole) of p-nitrobenzyl 7-[2-(2-thienylacetamido]-3-hydroxy-3-cephem-4-carboxylate in 50 ml. of dryN,N-dimethylacetamide were added 2 ml. of propylene oxide. To thesolution was added with stirring one equivalent of methanesulfonylchloride, and stirring was continued for 3 hours. The reaction mixturewas then taken up in ethyl acetate, and the solution was washed with asaturated solution of sodium chloride. The washed organic phase wasevaporated in vacuo to dryness to obtain the reaction product mixture asa residue. The reaction product was purified by preparative thin layerchromatography on silica gel using for elution 65 percent ethylacetate/hexane.

The purified product gave the following percent elemental composition onmicroanalysis.

Calculated for C₂₁ H₁₉ N₃ O₉ S₃ Theory: C, 45.56; H, 3.46; N, 7.59; S,17.38. Found: C, 45.74; H, 3.56; N, 7.30; S 17.06.

The nuclear magnetic resonance spectrum and the infrared absorptionsprectrum were in agreement with the structure of the title compound.

To 93 mg. of dicyclohexyl-18-crown -6 ether in 15 ml. of acetonitrile(dried over molecular sieves) were added 25 mg. of potassium fluoridewhich had been dried in vacuo at 90° C. The mixture was stirred for tenminutes, and 138 mg. of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-methylsulfonyloxy-3-cephem-4-carboxylate in4 ml. of acetonitrile were then added. The mixture was stirred for 1hour. The mixture was acidified by addition of dilute (5%) HCl, and theresulting acidified mixture was extracted with ethyl acetate. Pureproduct was obtained from the ethyl acetate extract by preparative thinlayer chromatography on silica gel using ethyl acetate:benzene (1:1).Ten mg. of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylate wereobtained.

I.R.: absorption peaks at 1792, 1740, and 1685 cm.⁻¹ NMR (CDCl₃) showedsignals at 6.15 (s, 2H, α-CH₂), 4.97 (d, 1H, J═4Hz, C₆ --H), 4.20 (q,1H, C₇ --H), 3.52 (d, 1H, C₇ --NH), 2.32-1.7 (m, 2H, C₂ --H₂) tau.Fluorine NMR -- (d, J═10 Hz). M.S.: calculated -- 477.0465 found --477.0455

Fragment ##STR46##

calculated -- 297.0345,

found -- 297.0344.

To 110 ml. of methanol and 83 mg. of pre-reduced 5 percent palladium oncarbon were added 83 mg. of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-cephem-4-carboxylate. The mixture washydrogenated at 53 psig. for one hour. The resulting mixture wasfiltered, the filtered catalyst was washed with methanol, and themethanol washings were added to the filtrate. The filtrate was thenevaporated in vacuo. The residue was dissolved in ethyl acetate, and theethyl acetate solution was extracted with dilute aqueous sodiumbicarbonate. The sodium bicarbonate solution was washed with ethylacetate, layered with ethyl acetate, and dilute aqueous HCl was added.The layered ethyl acetate was separated and evaporated to recover7-[2-(2-thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylic acid, shown bybioautogram to be biologically active.

EXAMPLE 28 p-Nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylatehydrochloride

To a solution of 500 mg. of p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate in 6 ml. ofmethylene chloride was added 95 mg. of dry pyridine and 237 mg. ofphosphorus pentachloride. The reaction mixture was stirred at roomtemperature for 1.5 hours, was thereafter cooled in an ice-water bath toabout 5° C. and 0.6 ml. of isobutyl alcohol were added. On continuedcooling and stirring the reaction product, p-nitrobenzyl7-amino-3-chloro-3-cephem-4-carboxylate hydrochloride, crystallized fromthe reaction mixture. The product was filtered, washed with coldmethylene chloride and dried to yield 200 mg. of the crystalline productmelting with decomposition at about 168° C.

Percent elemental composition for C₁₄ H₁₃ ClN₃ O₅ S.HCl: Theory: C,41.39; H, 3.20; N, 10.34; Cl, 17.45 Found: C, 41.14; H, 3.31; N, 10.44;Cl, 17.29.

I.R. (nujol mull): showed absorption bands, at 5.55 (β-lactam carbonyl)and, at 5.78 (ester carbonyl) microns.

UV (pH 7 buffer): showed absorption maximum, λ max 268 mμ (ε = 13,800).

N.M.R. (DMSO d₆): signals at 5.97 (s, 2H, C₂ --H₂), 4.8-4.5, (m, 4H, C₆--H, C₇ --H and ester CH₂), and 2.35-1.6, (q, 4H, aromatic H) tau.

The free base of the above nucleus ester hydrochloride salt, namely,p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate, gave thefollowing percent elemental composition on elemental analysis:

Calculated for C₁₄ H₁₂ ClN₃ O₅ S: Theory: C, 45.47; H, 3.27; N, 31,9.59, Found: C, 45.20; H, 3.48; N, Cl, 9.61.

EXAMPLE 29 7-Amino-3-chloro-3-cephem-4-carboxylic acid

To a solution of 750 mg. (1.85 mmole) of p-nitrobenzyl7-amino-3-chloro-3-cephem-4-carboxylate hydrochloride in 20 ml. oftetrahydrofuran and 40 ml. of methanol was added a suspension of 750 mg.of pre-reduced 5% palladium on carbon catalyst in 20 ml. of ethanol andthe suspension was hydrogenated under 50 psi of hydrogen at roomtemperature for 45 minutes. The catalyst was filtered and washed withTHF and water. The filtrate and catalyst washes were combined andevaporated to dryness. The residue was dissolved in a water-ethylacetate mixture and the pH adjusted to pH 3. The insoluble product wasfiltered and triturated with acetone. The product was then dried toyield 115 mg. of 7-amino-3-chloro-3-cephem-4-carboxylic acid.

I.R. mull): absorption peaks at, 5.61 (β-lactam carbonyl), and, 6.2(carboxylic acid).

NMR (D₂ O-NaHCO₃): signals at, 6.25 (ABq, 2H, C₂ --H₂) 4.88 (d, 1H, C₆--H) and 4.54 (d, 1H, C₇ --H) tau.

U.V. (pH 7 buffer): absorption maximum at λ max 265 mμ, ε = 7550

EXAMPLE 30 Diphenylmethyl 7-amino-3-chloro-3-cephem-4-carboxylate

To a solution of 525 mg. of diphenylmethyl7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate in 20 ml. ofmethylene chloride was added 0.1 ml. of dry pyridine and 237 mg. ofphosphorus pentachloride. The reaction mixture was stirred for 2 hoursat room temperature and was then cooled in an ice-water mixture. To thecold mixture was added 0.6 ml. of isobutanol and after 30 minutes thereaction mixture was evaporated. The residue was dissolved in ethylacetate and the solution was washed with 5% sodium bicarbonate and withwater and was dried. The dried solution was evaporated to dryness andthe residue was triturated with ether to yield 190 mg. of 3-chloronucleus ester, diphenylmethyl 7-amino-3--chloro-3-cephem-4-carboxylate.

I.R. (mull): absorption peaks at 5.7 and 5.9 (β-lactam and estercarbonyl) microns.

NMR (CDCl₃): signals at 6.35 (ABq, 2H, C₂ --H₂), 4.78 (2d, 2H, C₆ --H),3.05 (s, 1H, ester CH) and 2.65 (s, 10H, aromatic H).

EXAMPLE 31

Following the 7-acyl side chain cleavage reaction conditions describedin Example 30 p-nitrobenzyl 7-amino-3-fluoro-3-cephem-4-carboxylate wasprepared with the intermediate of Example 27, p-nitrobenzyl7-[2-(2-thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylate.

EXAMPLE 32

Following the 7-acyl side chain cleavage reaction procedure described byExample 30 diphenylmethyl 7-amino-3-bromo-3-cephem-4-carboxylate isprepared with diphenylmethyl7-phenoxyacetamido-3-bromo-3-cephem-4-carboxylate.

EXAMPLE 33 7-(D-Mandelamido)-3-chloro-3-cephem-4-carboxylic acid

To a suspension of 812 mg. (2mmole) of p-nitrobenzyl7-amino-3-chloro-3-cephem-4-carboxylic acid hydrochloride in 40 ml. ofethyl acetate was added a solution of 520 mg. (5 mmole) of sodiumbisulfite in 40 ml. of water. The mixture was vigorously stirred while395 mg. (2.2 mmole) of D-mandelic acid O-carboxy anhydride were added.The mixture was stirred for 1.5 hours at room temperature and theaqueous layer was separated from the ethyl acetate layer and washed withethyl acetate. The ethyl acetate wash was combined with the ethylacetate layer and the combined wash and ethyl acetate layer were washedseveral times with water and then dried and evaporated to yield thereaction product as a dry residue. The residue was triturated with etherto yield 685 mg. of p-nitrobenzyl7-(D-mandelamido)-3-chloro-3-chloro-3-cephem-4-carboxylate melting atabout 158°-164° C. with decomposition.

Elemental analysis for C₂₂ H₁₈ N₃ O₇ SCl: Theory: C, 52.44; H, 3.60; N,8.34; Cl, 7.04% Found: C, 52.25; H, 3.45; N, 8.58; Cl, 6.82%.

NMR (CDCl₃): signals at 6.24 (ABq, 2H, C₂ --H₂), 5.0-4.7, (m, 2H, C₆ --Hand α-H), 4.57 (s, 2H, ester CH₂), 6.23 (q, 1H, C₇ --H), and 2.8-1.2 (m,10H, aromatic H and C₇ --NH) tau.

UV (acetonitrile): λ max 265 mμ (ε = 18,600).

The reaction product, 200 mg., was reacted with hydrogen in the presenceof 5% palladium on carbon to effect removal of the p-nitrobenzyl estergroup and provide 75 mg. of7-(D-mandelamido)-3-chloro-3-cephem-4-carboxylic acid.

NMR (D₂ O-sodium bicarbonate): signals at 6.42 (ABq, 2H, C₂ --H₂), 4.90(d, 1H, C₆ --H), 4.68 (s, 1H, α-CH), 4.37 (d, 1H, C₇ --H) and 2.49 (s,5H, aromatic H)tau.

EXAMPLE 34 7-[0-(Formyl)-D-mandelamido]-3-chloro-3-cephem-4-carboxylicacid

p-Nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate, 2.2 g. (5.9mmole) was acylated with 1.08 mg. (6 mmole) of O-formyl mandelic acid indry tetrahydrofuran in the presence of 1.5 g. (6 mmole) of thecondensing agent E.E.D.Q. to provide 2 g. (65 percent yield) of theproduct p-nitrobenzyl7-[O-(formyl)D-mandelamido]-3-chloro-3-cephem-4-carboxylate. The productwas obtained crystalline from ethyl acetate.

Elemental analysis calculated for C₃₃ H₁₈ ClN₃ O₈ S: Theory: C, 51.93;H, 3.41; N, 7.90; Cl, 6.67 percent. Found: C, 52.13; H, 3.27; N, 7.88;Cl, 6.88 percent.

Nuclear magnetic resonance spectrum (CDCl₃) showed signals at 6.40 (ABq,2H, C₂ --H₂), 4.97 (d, 1H, C₆ --H), 4.62 (s, 2H, ester-CH₂), 4.20 (q,1H, C₇ --H), 3.75 (s, 1H, α-CH), and 2.75--1.72 (m, 1OH, aromatic H andO-CHO) tau.

The above acylated ester product was de-esterified by hydrogenolysis ofthe p-nitrobenzyl ester group over prereduced 5 percentpalladium-on-carbon catalyst by following the procedures described inthe preceding Examples. From 2.0 g. (3.76 mmole) of the ester 1.2 g. ofthe product 7-[O-(formyl)-D-mandelamido]-3-chloro-3-cephem-4-carboxylicacid, were obtained.

Electrometric titration (66 percent aqueous DMF). showed a titratablegroup having a pKa of 4.5. The apparent molecular weight was 405. Thecalculated molecular weight is 397.

U.S. (acetonitrile); λ max 265 mμ (ε=78000).

The nuclear magnetic resonance spectrum of the product run in deuterateddimethyl sulfoxide (DMSO d₆) was consistent with the structure of theproduct: ##STR47##

EXAMPLE 35

This example demonstrates the preparation of a 3-halo-3-cephem compoundwith a 3-exomethylenecepham compound without the isolation andpurification of the 3-hydroxy-3-cephem intermediate.

A solution of 1 g. (2 mmole) of p-nitrobenzyl7-phenoxyacetamido-3-exomethylenecepham-4-carboxylate in methylenechloride was cooled to -78° C. and was ozonized. The ozonide wasdecomposed with sulfur dioxide and the solvent evaporated under reductedpressure. The crude residue was comprised of about 85 percent of the3-hydroxy-3-cephem ester as estimated from the nmr spectrum of theresidue run in deuterated dimethylsulfoxide.

The crude 3-hydroxy-3-cephem ester was reacted in dimethylformamide withphosphorus pentachloride to provide 1.4 g. of p-nitrobenzyl7-phenoxyacetamido-3-chloro-3-cephem-4-carboxylate. The nmr spectrum ofthe product run in DMSO d₆ compared with that of the previously preparedcompound. The overall yield of the product as calculated from the3-exomethylenecepham ester was 46 percent.

I claim:
 1. A compound of the formula ##STR48## wherein R₁ is hydrogen,benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl,2,2,2-trichloroethyl, t-butyl or a pharmaceutically acceptable estergroup of the formula ##STR49## X is fluoro, chloro, bromo or iodo; andthe hydrochloride salts thereof.
 2. The compound of claim 1 wherein X ischloro.
 3. The compound of claim 2 said compound being7-amino-3-chloro-3-cephem-4-carboxylic acid.
 4. The compound of claim 2said compound being p-nitrobenzyl7-amino-3-chloro-3-cephem-4-carboxylate.
 5. The hydrochloride salt ofthe compound of claim
 4. 6. The compound of claim 2 said compound beingdiphenylmethyl 7-amino-3-chloro-3-cephem-4-carboxylate.
 7. The compoundof claim 1 wherein X is fluoro.
 8. The compound of claim 1 wherein X isbromo.